Resistant Candida Albicans Strains Research Articles

Antifungal activity’s study of the of new derivatives of pyrrolo[3,4-c]pyrazol-3-ones and pyrazol-3- carboxamides in the biofilm model of Candida spp.

The search for new antifungal drugs is current interest due to the wide spread of fungal infections. To simulate the antifungal effect of new promising compounds with high antifungal activity in planktonic culture in clinical practice, it is necessary to study their effect on the biofilm of micromycetes.Aim: To study the antifungal activity of new promising representatives of silver salts of pyrazoles and their condensed systems under conditions of biofilm formation.Material and Methods. To study the antifungal activity of silver salts I and II, the micromethod of two-fold serial dilutions was used. Activity against a typical and 14 clinical highly virulent isolates of C. albicans was studied. The study of the antimycotic activity of substances under biofilm conditions was carried out using resazurin to quantify the degree of biofilm formation. The minimum inhibitory concentrations for biofilms were calculated (sMIC50 - the concentration of the antifungal substance at which a 50% decrease in fluorescence is observed compared to the positive control).Results and Discussion. It was shown that the antifungal effect of the studied compounds in biofilm culture of clinical strains of Candida spp. is significantly lower than in planktonic culture. High antifungal activity of the silver salt of pyrazole-3- carboxamide in planktonic and biofilm cultures of resistant strains of Candida albicans, exceeding the effect of the reference drug fluconazole by 2.8–11.2 times, was revealed.Conclusion. A promising pyrazole-3-carboxamide derivative has been identified that effectively inhibits the growth of Candida albicans biofilms, which can be recommended for further study.

Heteroleptic cobalt complex augments antifungal activity with fluconazole and causes membrane disruption in Candida albicans.

Heteroleptic metal complexes containing CuII, CoII, and ZnII, incorporating curcumin and a Schiff base ligand (L), were synthesized and characterized, and their antifungal activity was evaluated. Their antifungal activities were investigated individually and in combination with fluconazole. Utilizing various analytical techniques such as UV-Vis, FT-IR, NMR, ESI-MS, TGA-DTG, elemental analyses, conductance, and magnetic susceptibility measurements, complex C1 ([Cu(Cur)LCl(H2O)]) was assigned a distorted octahedral geometry, while complexes C2 ([Co(Cur)LCl(H2O)]) and C3 ([Zn(Cur)LCl(H2O)]) were assigned octahedral geometries. Among these complexes, C2 exhibited the highest inhibitory activity against both FLC-susceptible and resistant strains of Candida albicans. Furthermore, C2 demonstrated candidicidal activity and synergistic interactions with fluconazole, effectively inhibiting the growth and survival of both FLC-resistant and FLC-sensitive C. albicans strains. The complex displayed a dose-dependent inhibition of drug efflux pumps in FLC-resistant C. albicans strains, indicating its potential to disrupt the cell membrane of these strains. The significant role of membrane efflux transporters in the development of antifungal drug resistance within Candida species has been extensively documented and our findings indicate that complex C2 specifically targets this crucial factor, thereby playing a pivotal role in mitigating drug resistance in C. albicans.

Antimicrobial and antibiofilm effects of shikonin with tea tree oil nanoemulsion against Candida albicans and Staphylococcus aureus

The current work aims to develop a shikonin and tea tree oil loaded nanoemulsion system stabilized by a mixture of GRAS grade surfactants (Tween 20 and monoolein) and a cosurfactant (Transcutol P). This system was designed to address the poor aqueous solubility and photostability issues of shikonin. The authenticity of shikonin employed in this study was confirmed using nuclear magnetic resonance (NMR) spectroscopy. The optimized nanoemulsion exhibited highly favorable characteristics in terms of zeta potential (−23.8 mV), polydispersity index (0.216) and particle size (22.97 nm). These findings were corroborated by transmission electron microscopy (TEM) micrographs which confirmed the spherical and uniform nature of the nanoemulsion globules. Moreover, attenuated total reflectance (ATR) and X-ray diffraction analysis (XRD) analysis affirmed improved chemical stability and amorphization, respectively. Photodegradation studies were performed by exposing pure shikonin and the developed nanoemulsion to ultraviolet light for 1 h using a UV lamp, followed by high performance liquid chromatography (HPLC) analysis. The results confirmed that the developed nanoemulsion system imparts photoprotection to pure shikonin in the encapsulated system. Furthermore, the research investigated the effect of the nanoemulsion on biofilms formed by Candida albicans and methicillin resistant Staphylococcus aureus (MRSA). Scanning electron microscopy, florescence microscopy and phase contrast microscopy unveiled a remarkable reduction in biofilm area, accompanied by disruptions in the cell wall and abnormalities on the cell surface of the tested microorganisms. In conclusion, the nanoencapsulation of shikonin with tea tree oil as the lipid phase showcased significantly enhanced antimicrobial and antibiofilm potential compared to pure shikonin against resistant strains of Candida albicans and Staphylococcus aureus.

Anti-Candida albicans Effects and Mechanisms of Theasaponin E1 and Assamsaponin A.

Candida albicans is an opportunistic human fungal pathogen, and its drug resistance is becoming a serious problem. Camellia sinensis seed saponins showed inhibitory effects on resistant Candida albicans strains, but the active components and mechanisms are unclear. In this study, the effects and mechanisms of two Camellia sinensis seed saponin monomers, theasaponin E1 (TE1) and assamsaponin A (ASA), on a resistant Candida albicans strain (ATCC 10231) were explored. The minimum inhibitory concentration and minimum fungicidal concentration of TE1 and ASA were equivalent. The time-kill curves showed that the fungicidal efficiency of ASA was higher than that of TE1. TE1 and ASA significantly increased the cell membrane permeability and disrupted the cell membrane integrity of C. albicans cells, probably by interacting with membrane-bound sterols. Moreover, TE1 and ASA induced the accumulation of intracellular ROS and decreased the mitochondrial membrane potential. Transcriptome and qRT-PCR analyses revealed that the differentially expressed genes were concentrated in the cell wall, plasma membrane, glycolysis, and ergosterol synthesis pathways. In conclusion, the antifungal mechanisms of TE1 and ASA included the interference with the biosynthesis of ergosterol in fungal cell membranes, damage to the mitochondria, and the regulation of energy metabolism and lipid metabolism. Tea seed saponins have the potential to be novel anti-Candida albicans agents.

Effect of new methionine-based ionic liquid on the CDR1 and CDR2 gene expression on sensitive and resistant strains of Candida albicans.

Previous researchers showed the antimicrobial ability of ionic liquids (ILs) on different infective agents. ILs can dissolve organic components, especially DNA molecules. Among synthesized eight binary ILs mixtures, we have chosen ([Met-HCl] [PyS]) IL for determining the antifungal ability of IL against Candida albicans cells. Well diffusion assay, chrome agar and Germ tube tests were used to detect the Candida samples. PCR, real-time-PCR, and flow cytometry tests were performed to determine the IL's rate of toxic ability. Well diffusion assay revealed the diameters of the growth inhibition zones were the largest in IL with methionine and Proline amino acids. Minimum inhibitory concentration (MIC) and the Minimum fungicidal concentration (MFC) tests showed that they inhibited the growth of the C. albicans at a range from 250 μg/ml for sensitivity and 400 μg/ml for resistance, MIC average of all samples were 341.62 ± 4.153 μg/ml. IL reduced the expression of CDR1 and CDR2 the genes encoded by the major protein of ABC system transporter by 2.1 (P= 0.009) and 1.2 fold (P= 0.693), revealed by PCR and real time-PCR. In the flow cytometry test, there were increasing dead cells after treating with the ([Met-HCl] [PyS]) even in the most resistant strain. The novel IL was effective against the most clinical and standard C. albicans.

Antagonist activities and phylogenetic relationships of actinomycetes isolated from an Artemisia habitat

Diverse habitats have been screened for novel antimicrobial actinomycetes, while others remain unexplored. In this study, we analyzed the bioactivities of actinomycetes cultured from rhizosphere soils of the desert plant Artemisia tridentata and the nearby bulk soils. Actinomycetes were screened for antifungal and antibacterial activities toward a panel of plant pathogens; all comparisons were between activities of rhizosphere soil isolates toward those of its counterpart bulk soil. A selected group of the strongest antifungal isolates were also tested against two antifungal-drug resistant strains of Candida albicans. 16S rDNA partial sequences and phylogenetic analysis of isolates that showed broad-spectrum antifungal activities were performed. Forty-two out of 200 and two soil isolated actinomycetes were selected for their strong antifungal activities. The highest proportion of isolates (p<0.05) from rhizosphere soil of an old plant showed antagonism against gram-positive bacteria (0.483 and 0.224 proportions against Bacillus subtilis and Rathayibacter tritici, respectively), and phytopathogenic fungi (0.259, 0.431, and 0.345 proportions against Fusarium oxysporum, Rhizoctonia solani and Pythium ultimum, respectively), while the highest antagonism against the gram-negative bacteria predominated in isolates from the bulk soils. Isolates from a rhizosphere soil of a young plant were characterized for strong antagonist activities against Fusarium oxysporum (0.333 proportion, p<0.05). Phylogenetic analysis of 16S rDNA sequences showed that isolates that exhibited strong antifungal activity were genetically similar. We conclude that the rhizosphere soil of A. tridentata is an excellent source for discovery of actinomycetes with potentially novel antifungal compounds.

Evaluation of the antifungal powers of five plant species of the genus Terminalia on strains responsible for candidiasis

Background: Candidiasis is a fungal disease caused by Candida albicans, a yeast that preferentially affects mucous membranes. These diseases are more and more recurrent because of the generalized decline in immunity and the appearance of resistance to classical antifungal drugs. Therefore, fighting against therapeutic failures becomes necessary. The objective of this study is to contribute to the fight against candidiasis through the research of new antifungal molecules. To do so, the hydroalcoholic extracts of Terminalia catappa, Terminalia glaucescens, Terminalia ivorensis, Terminalia mantaly and Terminalia superba, five plant species used in traditional medicine against dermatoses, were tested on the in vitro growth of resistant strains of Candida albicans. In addition, the polyphenol composition of these plants was carried out in order to evaluate their protective powers.&#x0D; Methods: The antifungal tests were performed by the solid-state dilution method. While the determination of polyphenols was performed by the photometric method.&#x0D; Results: The results showed an inhibition of the growth of germs by four plants except Terminalia catappa. The minimum inhibitory concentrations (MIC) obtained are as follows: MIC (T. ivorensis) =0.25 mg/mL; MIC (T. glaucescens) =0.50 mg/mL; MIC (T. superba) = 4 mg/ML; MIC (T. mantaly) = 2 mg/mL.&#x0D; Conclusion: This study indicates that T. ivorensis is the most active and has the highest content of phenolic compounds. This plant could be a source of effective molecules in the treatment of candidiasis.&#x0D; Keywords: Candida albicans, candidiasis, Terminalia, antifungal, polyphenol,

Inhibitory effects of different fractions separated from standardized extract of Myrtus communis L. against nystatin-susceptible and nystatin-resistant Candida albicans isolated from HIV positive patients

Backgroundand Purpose: Myrtle (Myrtus communis L.) is a medicinal herb that plays an essential role in treating fungal infections. The present study investigated the antifungal properties of different fractions of the M. communis L. leaf extract against Candida albicans (susceptible and resistant to nystatin). Materials and methodsTotal extract (TE) and petroleum ether (PE), chloroform (CH), ethyl acetate (EA), and methanol (ME) fractions were prepared using the sonication method. The study used the standard strain sample (ATCC 76645) and nystatin-resistant C. albicans from oral samples of HIV-infected individuals. The identification of resistant isolate was performed using phenotypic and molecular methods. Minimum inhibitory concentration (MIC) and Minimum fungicidal concentration (MFC) of the fractions along total extract were determined by microdilution method on nystatin-resistant and susceptible Candida albicans. The Folin-Ciocalteu method was used to determine the total phenolic content of the extract. ResultsThe extraction yield (w/w) was 13.50 for TE, 2.10 for PE, 2.23 for Ch, 2.14 for EA, and 10.03 for ME fractions. Chloroform extract showed good anti-candida activity against nystatin susceptible and resistant C. albicans (62.5 ​μg/mL). Ethyl acetate fraction exhibited the greatest MIC against nystatin susceptible and resistant C. albicans (250 ​μg/mL). The MIC value of fluconazole was >64 μg/mL for both susceptible and -resistant strains. The amount of phenolic compounds of the total extract was reported to be equal to 5.4%, equivalent to gallic acid. ConclusionResults revealed that the PE and CH fractions showed greater antifungal effects than the total extract against both susceptible and resistant strains of Candida albicans. It can conclude that active antifungal compounds of the plant belong to a specific group of metabolites, which according to the type of solvent, probably have non-polar nature. Further separation is carrying out.

A novel CRISPRi platform to study the role of essential genes in antifungal drug-resistant Candida albicans isolates

With the emergence of antifungal resistant Candida albicans strains, the need for new antifungal drugs is critical in combating this fungal pathogen. Investigating essential genes in C. albicans is a vital step in characterizing putative antifungal drug targets. As some of these essential genes are conserved between fungal organisms, developed therapies targeting these genes have the potential to be broad range antifungals. In order to study these essential genes, classical genetic knockout or CRISPR-based approaches cannot be used as disrupting essential genes leads to lethality in the organism. Fortunately, a variation of the CRISPR system (CRISPR interference or CRISPRi) exists that enables precise transcriptional repression of the gene of interest without introducing genetic mutations. CRISPRi utilizes an endonuclease dead Cas9 protein that can be targeted to a precise location but lacks the ability to create a double-stranded break. The binding of the dCas9 protein to DNA prevents the binding of RNA polymerase to the promoter through steric hindrance thereby reducing expression. We recently published the novel use of this technology in C. albicans and are currently working on expanding this technology to large scale repression of essential genes. Through the construction of an essential gene CRISPRi-sgRNA library, we can begin to study the function of essential genes under different conditions and identify genes that are involved in critical processes such as drug tolerance in antifungal resistant background strains. These genes can ultimately be characterized as putative targets for novel antifungal drug development, or targeted as a means to sensitize drug-resistant strains to antifungal treatment.

Fluconazole nanosuspension enhances in vitro antifungal activity against resistant strains of Candida albicans

Background: Recently, nanoparticles were widely used in drug delivery. Fluconazole (FLZ) is a lipid-soluble antifungal, which is utilized in treating fungal infections. The current work aimed to investigate the characteristics and antifungal activity of FLZ nanosuspension. Methods: FLZ nanosuspensions were prepared by Ultrasonication (simple and containing polymer). Surfactants in various concentrations were dissolved in the deionized water, and the drug powder was dispersed in the surfactant solution by a high-speed homogenizer to achieve nanosuspension. The polymer was added to the selected formula. FLZ nanosuspension characteristics, including polydispersity, mean particle size, entrapment efficacy, and zeta potential, were assessed. The release profile via dialysis membrane, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (ATR-FTIR), and TEM were performed for nanosuspension evaluation. Antifungal activity against resistant strains of C. albicans was defined according to the CLSI document guideline. To analyze the results, one-way ANOVA was used, followed by Tukey test. Results: The results showed that increased sonication time and hydrophilic-lipophilic balance (HLB) significantly affected particle size reduction. Moreover, modification in the formulation components had a significant effect on the drug release process, furthermore affecting the properties of the nanoparticles. ATR-FTIR showed no chemical interaction between FLZ and formulation components. Compare to FLZ, a significant reduction (p &lt; 0.05) was detected in the MIC values of both FLZ-resistant and FLZ-susceptible strains of C. albicans against FLZ nanosuspension. Conclusion: It can be concluded that the ratio and amount of surfactants, the HLB, and the sonication process have effects on the properties of the nanoparticle’s characteristics, and selected nanoparticles show suitable antifungal effect against resistant strains of C. albicans.

Skirmishing MDR strain of Candida albicans by effective antifungal CeO2 nanostructures using Aspergillus terreus and Talaromyces purpurogenus

Emerging antibiotics resistance fungal infectionsis a major global health problem and new antifungal formulations are direly needed to fight drug resistant Candida albicans strains. This study is aimed to synthesize effective antifungal nanostructures of cerium oxide (CeO2) using culture filtrates of two common fungal strains Aspergillus terreus and Talaromyces pupureogenus. The fungal strains used in the synthesis were identified by 18S rRNA gene sequencing and deposited to NCBI GenBank with the accession number of MN099077 and MN121629, respectively. The biofabricated CeO2 NPs were characterized by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). Pure CeO2 nanoparticles (NPs) synthesized using Aspergillus terreus culture filtrate were depicted spherical morphology with average size of 28.5 nm. The CeO2NPs synthesized using Talaromyces pupureogenus revealed the presence of nanosponges with average size of 21.4 nm. Gas chromatography mass spectrometry of culture filtrates of respective strains indicated the presence of ethanol, 1-propanol and tri-chloromethane in culture filtrate of Aspergillus terreus and with addition of palmitic acid in Talaromyces pupureogenus culture filtrate which may have a function as bio reducers and capping agents. Dose dependent anticandidal activity of CeO2 NPs using various different concentrations (100, 200, 300, 600 μg ml−1) synthesized by both fungal strains was observed by disc diffusion assay against Candida albicansas evidenced by increase in size of zone of inhibitions with increasing concentration of CeO2NPs. Further in-vitro and in-vivo experiments are required to access the potential of CeO2 NPs for controlling Candida albicans strains.

Thymus vulgaris essential oil and thymol inhibit biofilms and interact synergistically with antifungal drugs against drug resistant strains of Candida albicans and Candida tropicalis

Role of biofilm in disease development and enhance tolerance to antifungal drugs among Candida species has necessitated search for new anti-fungal treatment strategy. Interference in pathogenic biofilm development by new antifungal compounds is considered as an attractive anti-infective strategy. Therefore, the objective of this study was to evaluate Thymus vulgaris essential oil and its major active compound, thymol for their potential to inhibit and eradicate biofilms alone and in combination with antifungal drugs against Candida spp. with especial reference to Candida tropicalis. Anti-candidal efficacy of T. vulgaris and thymol in terms of minimum inhibitory concentration (MIC) was first determined to select the sub-MICs against C. albicans and C. tropicalis. Biofilm formation in the presence and absence of test agents was determined in 96-well microtiter plate by XTT reduction assay and effect of essential oils at sub-MICs of the test agents on biofilm development on glass surface was analysed by light and scanning electron microscopy. Synergistic interaction between essential oils and antifungal drugs were studied by checkerboard method. Effect of sub-MIC of T. vulgaris (0.5×MIC) and thymol (0.5×MIC) on biofilm formation showed a significant reduction (P<0.05) in biofilms. Light microscopy and SEM studies revealed disaggregation and deformed shape of C. albicans biofilm cells and reduced hyphae formation in C. tropicalis biofilm cells at sub-MICs of thymol. Significant effect of T. vulgaris and thymol was also recorded on pre-formed biofilms of both C. albicans and C. tropicalis. T. vulgaris and thymol also showed synergy with fluconazole against both in planktonic and biofilm mode of growth of C. albicans and C. tropicalis. However, synergy with amphotericin B is clearly evident only in planktonic Candida cells. Thyme oil and thymol alone or in combination with antifungal drugs can act as promising antibiofilm agent against drug resistant strains of Candida species and needs further in vivo study to synergise its therapeutic efficacy.

Investigation of Mutations of ERG11 Gene in Fluconazole Resistant Strains of Candida Albicans Isolated From Patients With Volvovaginitis in West of Mazandaran

Investigation of Mutations of ERG11 Gene in Fluconazole Resistant Strains of Candida Albicans Isolated From Patients With Volvovaginitis in West of Mazandaran

The Synergistic Effect of Azoles and Fluoxetine against Resistant Candida albicans Strains Is Attributed to Attenuating Fungal Virulence.

This study evaluated the synergistic effects of the selective serotonin reuptake inhibitor, fluoxetine, in combination with azoles against Candida albicans both in vitro and in vivo and explored the underlying mechanism. MICs, sessile MICs, and time-kill curves were determined for resistant C. albicans Galleria mellonella was used as a nonvertebrate model for determining the efficacy of the drug combinations against C. albicans in vivo For the mechanism study, gene expression levels of the SAP gene family were determined by reverse transcription (RT)-PCR, and extracellular phospholipase activities were detected in vitro by the egg yolk agar method. The combinations resulted in synergistic activity against C. albicans strains, but the same effect was not found for the non-albicans Candida strains. For the biofilms formed over 4, 8, and 12 h, synergism was seen for the combination of fluconazole and fluoxetine. In addition, the time-kill curves confirmed the synergism dynamically. The results of the G. mellonella studies agreed with the in vitro analysis. In the mechanism study, we observed that fluconazole plus fluoxetine caused downregulation of the gene expression levels of SAP1 to SAP4 and weakened the extracellular phospholipase activities of resistant C. albicans The combinations of azoles and fluoxetine showed synergistic effects against resistant C. albicans may diminish the virulence properties of C. albicans.

Synergism Effect of the Essential Oil from Ocimum basilicum var. Maria Bonita and Its Major Components with Fluconazole and Its Influence on Ergosterol Biosynthesis.

The aim of this study was to evaluate the activity of the EO and its major components of Ocimum basilicum var. Maria Bonita, a genetically improved cultivar, against the fluconazole sensitive and resistant strains of Candida albicans and Cryptococcus neoformans. Geraniol presented better results than the EO, with a low MIC (76 μg/mL against C. neoformans and 152 μg/mL against both Candida strains). The combination of EO, linalool, or geraniol with fluconazole enhanced their antifungal activity, especially against the resistant strain (MIC reduced to 156, 197, and 38 μg/mL, resp.). The ergosterol assay showed that subinhibitory concentrations of the substances were able to reduce the amount of sterol extracted. The substances tested were able to reduce the capsule size which suggests they have an important mechanism of action. Transmission electron microscopy demonstrated cell wall destruction of C. neoformans after treatment with subinhibitory concentrations. In C. albicans ultrastructure alterations such as irregularities in the membrane, presence of vesicles, and cell wall thickening were observed. The biofilm formation was inhibited in both C. albicans strains at MIC and twice MIC. These results provide further support for the use of O. basilicum EO and its major components as a potential source of antifungal agents.

In vitro antifungal activity of essential oils from local plants against fluconazole-resistant oral Candida albicans isolates

Background and objective: Candida albicans is an oral commensal flora that causes opportunistic local and systemic infections in immunocompromied individuals. Fluconazole is frequently used for treating patients with active infections or preventing recurrent infections. The emergence of resistant strains encouraged scientists to search for compounds that have antifungal property and can overcome the usual microbial resistant mechanisms to antimicrobial agents. Essential oils from natural plants have received great interest due to the antimicrobial property of their multiple constituents. The aim of this study was to investigate the antifungal activity of Rosemary and Thyme essential oils against fluconazole resistant oral Candida albicans isolates. Methods: Essential oil from areal parts of Rosemarium officinalis and Thymus vulgaris were obtained by hydrodistillation. Disk diffusion and microbroth dilution methods were followed to test the sensitivity of eight fluconazole resistant oral Candida albicans isolates and one ATCC strain to the extracted Essential oils. Minimum inhibitory concentration and minimum fungicidal concentration were used to evaluate the antifungal potential of the tested essential oils. Results: The results of disk diffusion method using concentrated Essential oils showed marked inhibition of growth around the prepared discs from both Essential oils. However, the recoded diameters was higher in Thyme Essential oil (Mean ± SD: 42.4 ± 6.5) when compared with Rosemary essential oil (Mean ± SD: 11.8 ± 2.8). Serial two fold dilutions of the tested essential oils showed that both essential oils attain their antifungal activities even at very low concentrations. The highest MIC and MFC of Rosemary EO were 3.125% and 6.25%, respectively. Thyme essential oil showed very low MIC and MFC (<1.56%). Conclusion: Thyme and Rosemary essential oils were ideal natural compounds against drug resistant Candida albicans strains.

Chemosensitization of multidrug resistant Candida albicans by the oxathiolone fused chalcone derivatives.

Three structurally related oxathiolone fused chalcone derivatives appeared effective chemosensitizers, able to restore in part sensitivity to fluconazole of multidrug-resistant C. albicans strains. Compound 21 effectively chemosensitized cells resistant due to the overexpression of the MDR1 gene, compound 6 reduced resistance of cells overexpressing the ABC-type drug transporters CDR1/CDR2 and derivative 18 partially reversed fluconazole resistance mediated by both types of yeast drug efflux pumps. The observed effect of sensitization of resistant strains of Candida albicans to fluconazole activity in the presence of active compounds most likely resulted from inhibition of the pump-mediated efflux, as was revealed by the results of studies involving the fluorescent probes, Nile Red, Rhodamine 6G and diS-C3(3).

Antifungal activity of azole compounds CPA18 and CPA109 against azole-susceptible and -resistant strains of Candida albicans

In this study we investigated the in vitro fungistatic and fungicidal activities of CPA18 and CPA109, two azole compounds with original structural features, alone and in combination with fluconazole against fluconazole-susceptible and -resistant Candida albicans strains. Antifungal activities were measured by MIC evaluation and time-kill studies. Azole binding analysis was performed by UV-Vis spectroscopy. Hyphal growth inhibition and filipin and propidium iodide staining assays were used for morphological analysis. An analysis of membrane lipids was also performed to gauge alterations in membrane composition and integrity. Synergism was calculated using fractional inhibitory concentration indices (FICIs). Evaluation of cytotoxicity towards murine macrophages was performed to verify selective antifungal activity. Even though their binding affinity to C. albicans Erg11p is comparable to that of fluconazole, CPA compounds are active against resistant strains of C. albicans with a mutation in ERG11 sequences and/or overexpressing the ABC transporter genes CDR1 and CDR2, which encode ATP-dependent efflux pumps. Moreover, CPA18 is fungistatic, even against the two resistant strains, and was found to be synergistic with fluconazole. Differently from fluconazole and other related azoles, CPA compounds induced marked changes in membrane permeability and dramatic alterations in membrane lipid composition. Our outcomes suggest that CPA compounds are able to overcome major mechanisms of resistance in C. albicans. Also, they are promising candidates for combination treatment that could reduce the toxicity caused by high fluconazole doses, particularly in immunocompromised patients.

Antifungi Efficacy of the Compound Preparation of &lt;i&gt;Anemarrhena asphodeloides&lt;/i&gt; Bunge on the Drug Resistant Strain of Candida Albicans

To investigate the efficacy of the compound preparation named KZY-2 which include the effective group formula of anemarrhena asphodeloides bunge and rhizoma coptidis on the fluconazole and ifraconazo resistant strains of Candida albicans respectively in vitro. The fluconazole and ifraconazo resistant strains of Candida albicans were sieved by drug sensitive scrip test and the diameter of inhibition zone was measured adopting the method of agar plate diffusion, reference to the M27-A recommended by the United States National Laboratory Standards Committee (NCCLS), the minimal inhibitive concentration(MIC,mg/m1) of KZY-2 against the the standard strain(ATCC32354) and the fluconazole and ifraconazo resistant strains of candida albicans was measured respectively. Results showed that the 12 fluconazole and ifraconazo resistant strains which named RC1 to RC12 were sieved from the 20 strains Candida albicans isolated from clinic, the diameters of inhibited zone of KZY-2 against ATCC32354 was 32mm and against RC1～RC12 was between 12mm and 28mm, the MIC of KZY-2 against ATCC32354 was 312.5μg/m1 and against RC1～RC12 was between 625μg /m1 and 5000μg /m1. These results indicate that KZY-2 has good antifungi efficacy on the standard strain and the fluconazole and ifraconazo resistant strains of Candida albicans in vitro.

Detection of TAC1 gene point mutations in fiuconazole-resistant Candida albicans isolates with rolling circle amplification.

Objective To detect point mutations of TAC1 gene in fluconazole-resistant Candida albi-cans isolates with rolling circle amplification (RCA), develop an accurate, rapid and specific assay to detect single nucleotide polymorphisms (SNPs), and to estimate the relationship between mutations of TAC1 gene and resistance to fluconazole. Methods A total of 33 fluconazole-resistant Candida albicana isolates, including 8 strains from America and 25 from Australia, were collected. Four TAC1-specific padlock probes were designed according to previously reported mutations. DNA was extracted from these tested strains, subjected to amplification of three targeted fragments of TAC1 gene with PCR. Then, RCA was performed to detect point mutations of TAC1 gene in resistant Candida albicans strains. At the same time, the target fragments underwent sequencing analysis, and the results of RCA were compared with those of sequencing. Results Two types of resistance-associated mutations were found in 5 out of the 33 fluconazole-resistant strains. Among the 5 strains, 4 were from America, 1 harbored T225A mutation and 4 carried A736V mutation. No related mutation was found in TAC1 gene of 4 fluconazole-sensitive isolates. Conclusions RCA assay could accurately and rapidly detect point mutations of genes. Further studies are required to clarify the relationship between TAC1 point mutations and fluconazole resistance. Key words: Candida albicans; Fluconazole; Drug resistance, fungal; Point mutation; Rolling circle amplification