Cellular apoptosis and cell cycle arrest as potential therapeutic targets for eugenol derivatives in Candida auris.

Candida auris, the youngest Candida species, is known to cause candidiasis and candidemia in humans and has been related to several hospital outbreaks. Moreover, Candida auris infections are largely resistant to the antifungal drugs currently in clinical use, necessitating the development of novel medications and approaches to treat such infections. Following up on our previous studies that demonstrated eugenol tosylate congeners (ETCs) to have antifungal activity, several ETCs (C1-C6) were synthesized to find a lead molecule with the requisite antifungal activity against C. auris. Preliminary tests, including broth microdilution and the MUSE cell viability assay, identified C5 as the most active derivative, with a MIC value of 0.98 g/mL against all strains tested. Cell count and viability assays further validated the fungicidal activity of C5. Apoptotic indicators, such as phosphatidylserine externalization, DNA fragmentation, mitochondrial depolarization, decreased cytochrome c and oxidase activity and cell death confirmed that C5 caused apoptosis in C. auris isolates. The low cytotoxicity of C5 further confirmed the safety of using this derivative in future studies. To support the conclusions drawn in this investigation, additional in vivo experiments demonstrating the antifungal activity of this lead compound in animal models will be needed.

Dodonaea viscosa var angustifolia derived 5,6,8-trihydroxy-7,4' dimethoxy flavone inhibits ergosterol synthesis and the production of hyphae and biofilm in Candida albicans.

ETHNOPHARMACOLOGICAL RELEVANCE: Candida albicans is developing resistance to existing drugs increasing morbidity and mortality, which elevates an immediate need to explore new antifungal agents. Phytochemicals are an excellent source of therapeutic agents. We previously reported the antifungal activity of the crude extract of Dodonaea viscosa var. angustifolia Jacq. (DVA) from which a beneficial compound flavone: 5,6,8-trihydroxy-7,4' dimethoxy flavone (5,6,8-trihydroxy-7-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) abbreviated as TMMC, was extracted. AIM OF THE STUDY: The present study evaluated the effect of a TMMC subfraction on biofilms, membrane stability, ergosterol biosynthesis and germ tube (GT) formation in Candida albicans. MATERIALS AND METHODS: Extracts were prepared and fractionated to obtain purified TMMC. Minimum inhibitory concentrations of TMMC were obtained and subinhibitory concentrations were selected for further studies. Confocal laser scanning microscopy (CLSM) was performed to assess the effect of TMMC on membrane permeability and sterol deposition using propidium iodide (PI) and filipin stains, respectively. RESULTS: Minimum inhibitory concentrations (MIC) and Minimum Fungicidal concentrations (MFC) of TMMC were 0.39 mg/mL and 1.56 mg/mL, respectively. TMMC inhibited biofilm formation and damaged mature biofilms at 0.39 mg/mL and 1.56 mg/mL, respectively. CLSM further confirmed the disruption and architectural changes in biofilms following treatment with TMMC. TMMC also inhibited GT formation and ergosterol biosynthesis in a concentration dependent manner, which was further confirmed by varying sterol distribution and membrane disruption in treated and untreated cells. CONCLUSIONS: With the multiple targets at different concentrations, TMMC warrants its potential use as antifungal drug against C. albicans. However further studies using animal models and more mechanistic approaches will be required.

Cellular apoptosis and necrosis as therapeutic targets for novel Eugenol Tosylate Congeners against Candida albicans.

Despite the rise of new Candida species, Candida albicans tops the list with high morbidity and mortality rates. To tackle this problem there is a need to explore new antifungals that could replace or augment the current treatment options. We previously reported that tosylation of eugenol on hydroxyl group resulted in molecules with enhanced antifungal potency. In line with that work, we synthesized new eugenol tosylate congeners (ETC-1-ETC-7) with different substituents on pendent sulfonyl group and tested their susceptibility against different fluconazole susceptible and resistant C. albicans strains. We evaluated physiology and mode of cell death in response to the most active derivatives by analyzing major apoptotic markers in yeast such as phosphatidylserine externalization, DNA fragmentation, mitochondrial depolarization and decrease in cytochrome c oxidase activity. The results demonstrated that all C. albicans strains were variably susceptible to the test compounds with MIC ranging from 0.125-512 µg/ml, and the most active compounds (ETC-5, ETC-6 and ETC-7) actuate apoptosis and necrosis in Candida cells in a dose-dependent manner via metacaspase-dependent pathway. Furthermore haemolytic assay showed low cytotoxicity effect of these ETCs. Overall the results indicated that ETCs exhibit potential antifungal activity against C. albicans by activating apoptotic and necrotic pathways.

Improved efficacy of antifungal drugs in combination with monoterpene phenols against Candida auris.

Emergence of Candida auris has been described as a global health threat due to its ability to cause invasive infections with high mortality rate and multidrug resistance. Novel drugs and therapies are required to target this organism and its pathogenicity. Anti-virulence approach and combination therapy have been proposed as alternatives in recent years. This study evaluated the virulence factors in C. auris, combination antifungal activity of phenolic compounds with antifungal drugs and determined effect of the most active compound on positive pathogenicity markers of C. auris. Antifungal susceptibility profile of 25 clinical isolates of C. auris against antifungal agents as well as against phenolic compounds was obtained using CLSI guidelines. Combination of the most active phenolic compound with antifungal drugs was determined. Effect of carvacrol on the virulence factors was also studied. Carvacrol was the most active phenol with median MIC of 125 µg/ml and its combination with fluconazole, amphotericin B, nystatin and caspofungin resulted synergistic and additive effects in 68%, 64%, 96% and 28%, respectively. Combination also reduced the MIC values of the drugs. All test strains showed adherence ability to epithelial cells and 96% of strains produced proteinase. None of the strains produced hyphae and phospholipase. At low concentrations, carvacrol significantly inhibited the adherence ability and proteinase production (both p < 0.01). Carvacrol has antifungal and anti-virulence activity against C. auris. It also showed an enhanced antifungal activity in combination with antifungal agents. Therefore it has potential to be developed into a novel antifungal agent.

Imidazole clubbed 1,3,4-oxadiazole derivatives as potential antifungal agents.

A series of compounds in which 2-(4-ethyl-2-pyridyl)-1H-imidazole was clubbed with substituted 1,3,4-oxadiazole was synthesized and subjected to antifungal activity evaluation. In vitro assays indicated that several clubbed derivatives had excellent antifungal activity against different strains of laboratory and clinically isolated Candida species. Structural Activity Relationship (SAR) studies revealed that the presence and position of substituents on the phenyl ring of the 1,3,4-oxadiazole unit, guides the antifungal potential of the compounds, where compound 4b, 4c and 4g were found to be active against all the tested fungal strains. Impairment of ergosterol biosynthesis upon the concomitant treatment of 4b, 4c and 4g, revealed the possible mechanisms of antifungal action of these compounds. Inhibitors snugly fitting the active site of the target enzyme, as revealed by molecular docking studies, may well explain their excellent inhibitory activity.

The in vitro antimicrobial activity of Cymbopogon essential oil (lemon grass) and its interaction with silver ions.

BACKGROUND: It is well known that Cymbopogon (lemon grass) essential oil exhibits antimicrobial activity while the efficacy of silver ions as a disinfectant is equally well reported. HYPOTHESIS: The antimicrobial activity of CEO and Ag(+) and their synergistic combinations will be useful in improving the current treatment strategies for various infections. STUDY DESIGN: In the present study, we determined the chemical composition and in vitro antimicrobial activity of six different Cymbopogon essential oils (CEO's) alone and in combination with silver ions (Ag(+)) against two Gram-positive (Staphylococcus aureus and Enterococcus faecalis), two Gram-negative (Escherichia coli and Moraxella catarrhalis) and two yeast species (Candida albicans and Candida tropicalis). The nature of potential interactions was determined by fractional inhibitory concentration indices (FICIs) for CEO's and Ag(+) calculated from microdilution assays and time-kill curves. RESULTS: Gas chromatography-mass spectrometry results confirmed the presence of nerol, geranial and geraniol as major volatile compounds. Minimum inhibitory concentration (MIC) values confirmed that all the tested pathogens are variably susceptible to both CEO's as well as Ag(+). The MIC of CEO's and Ag(+) against all the tested pathogens ranged from 0.032 mg/ml to 1 mg/ml and 0.004 and 0.064 mg/ml respectively, whereas when assayed in combination the FICI values were drastically reduced to range between 0.258 and 2.186, indicating synergy, additive and indifferent interactions. The most prominent interaction was observed between Cymbopogon flexuosus essential oil and Ag(+) against C. albicans with ∑FIC = 0.254. The synergistic interactions were further confirmed through the construction of isobolograms and time-kill plots. Transmission electron microscopy showed disturbance in the cell envelope upon the concomitant treatment of CEO's and Ag(+), which ultimately leads to cell death. CONCLUSION: Results suggest that CEO's and Ag(+) when used in combination offers an opportunity to the formulation scientist to produce novel combinations acting synergistically in the continued quest to control important infectious pathogens.

Unravelling the complex antimicrobial interactions of essential oils--the case of Thymus vulgaris (thyme).

Thymus vulgaris has gained tremendous popularity as an ornamental, culinary herb and its use in phytotherapy is well established and supported in the literature. The objective of this study was to explore possible interactions between selected molecules within Thymus vulgaris essential oil (TvEO) to gain a better understanding of how this complex essential oil exerts its antimicrobial activity. Evaluation of the antimicrobial efficacy and interactions were assessed on the essential oil and volatile constituents against various pathogens. Interactions between molecules at various ratios were graphically observed through the construction of isobolograms. Gas chromatography-mass spectrometry (GC-MS) analysis revealed 22 compounds which collectively represent >95% of the oil composition. Based on their minimum inhibitory concentration (MIC) values, they were categorised into weak (≥4 mg mL⁻¹), moderate (2-4 mg mL⁻¹) and noteworthy active (≤2 mg mL⁻¹) compounds. For the combination study, 21% synergistic, 42% additive, 36% indifferent and 1% antagonistic interactions were observed. Most of the interactions were observed between the weak and highly active molecules, and interestingly, no synergistic interaction was observed between the highly active compounds. Synergistic and additive interactions between the strong and weaker antimicrobial constituents present in TvEO enhance the antimicrobial efficacy of this commercially important essential oil.

Ocimum sanctum essential oil inhibits virulence attributes in Candida albicans.

Candida albicans is an opportunistic human fungal pathogen which causes disease mainly in immunocompromised patients. Activity of hydrolytic enzymes is essential for virulence of C. albicans and so is the capacity of these cells to undergo transition from yeast to mycelial form of growth. Ocimum sanctum is cultivated worldwide for its essential oil which exhibits medicinal properties. This work evaluates the anti-virulence activity of O. sanctum essential oil (OSEO) on 22 strains of C. albicans (including a standard strain ATCC 90028) isolated from both HIV positive and HIV negative patients. Candida isolates were exposed to sub-MICs of OSEO. In vitro secretion of proteinases and phospholipases was evaluated by plate assay containing BSA and egg yolk respectively. Morphological transition from yeast to filamentous form was monitored microscopically in LSM. For genetic analysis, respective genes associated with morphological transition (HWP1), proteinase (SAP1) and phospholipase (PLB2) were also investigated by Real Time PCR (qRT-PCR). Results were analyzed using Student's t-test. OSEO inhibits morphological transition in C. albicans and had a significant inhibitory effect on extracellular secretion of proteinases and phospholipases. Expression profile of respective selected genes associated with C. albicans virulence by qRT-PCR showed a reduced expression of HWP1, SAP1 and PLB2 genes in cells treated with sub-inhibitory concentrations of OSEO. This work suggests that OSEO inhibits morphological transition in C. albicans and decreases the secretion of hydrolytic enzymes involved in the early stage of infection as well as down regulates the associated genes. Further studies will assess the clinical application of OSEO and its constituents in the treatment of fungal infections.

Composition of Cassia fistula oil and its antifungal activity by disrupting ergosterol biosynthesis.

Cassia fistula oil was investigated for antifungal activities against standard and clinical isolates of Candida species. Gas chromatography coupled with mass spectrometric (GC-MS) analysis of C. fistula oil revealed the presence of antimicrobial compounds like beta-sitosterol, stigmasterol, ergosterol, betulinic acid, lupeol, fucosterol, alpha-amyrin and friedelin. The minimum inhibitory concentration (MIC) of the pulp and seed oils ranged between 250-300 and 350-500 microg/mL respectively. Both oils also inhibited by > or = 63.8% ergosterol bio-synthesis in Candida cell wall {fluconazole (standard) > or = 89.1%)}. The MICs were significantly correlated with the ergosterol content decrease in the cell wall (Student's t test p < or = 0.005). We can, therefore, conclude that active compounds are present in Cassia fistula oil that primarily target ergosterol biosynthesis in Candida cell wall.

Antimicrobial activities of essential oil and methanol extract of Coriaria nepalensis.

Plant extracts and products have been used for centuries in traditional medicine; for most of them, in addition to the scant scientific credibility, the chemical composition and spectrum of activity are yet to be explored. To put forward this effort and to identify novel antimicrobial agents, the inhibitory activities of methanolic extract and essential oil from Coriaria nepalensis against various microorganisms including pathogenic yeast, and Gram-positive and negative bacteria were evaluated. Chemical compositions of C. nepalensis methanolic extract and essential oil were analysed by gas chromatography-mass spectrometry. In vitro susceptibility tests against all the tested isolates were performed in terms of minimum inhibitory concentration (MIC), and well diffusion assay using standard protocols. All microorganisms tested were profoundly found susceptible to both the C. nepalensis extract and oil with MIC values of 1.3-2.1 mg mL⁻¹ (Gram-positive bacteria), 1.4-2.2 mg mL⁻¹ (Gram-negative bacteria) and 0.9-1.6 mg mL⁻¹ (yeasts). The extent of inhibition was shown more by methanolic extract than by essential oil. This study is the first to report the antimicrobial activity of extracts obtained from the C. nepalensis. It can be concluded that the observed antimicrobial characteristics of C. nepalensis indicate that it might be a promising antimicrobial agent.

Antifungal activity of Coriaria nepalensis essential oil by disrupting ergosterol biosynthesis and membrane integrity against Candida.

Fungal diseases in humans have increased significantly with the advent of an expanding population of immunosuppressed patients and with the introduction of sophisticated life-saving medical procedures. Plant extracts and products have been used in traditional medicine for centuries. Coriaria nepalensis essential oil (CNEO) is known to possess antimicrobial activity. This study was an attempt to examine CNEO against various fluconazole-sensitive and -resistant Candida isolates. Insight into the mechanism of action was elucidated by flow cytometric analysis and ergosterol biosynthesis studies. The susceptibility tests for CNEO were carried out in terms of MIC and by disc diffusion assays against all Candida isolates, employing standard protocols. Insight into the mechanism of action was elucidated by propidium iodide cell sorting (FACS) and by assessing ergosterol content in treated and untreated isolates with the test entity. CNEO was found effective against all Candida isolates, including the resistant strains. While CNEO inflicts fungal cell death by disrupting membrane integrity, significant impairment of ergosterol biosynthesis was induced by the test entity. CNEO showed a strong antifungal effect against all the Candida isolates. Mechanisms of action appear to originate from the inhibition of ergosterol biosynthesis and the disruption of membrane integrity. It can be concluded that the observed antimicrobial characteristics of C. nepalensis indicate that it might be a promising antimicrobial agent.

Effect of garlic-derived allyl sulphides on morphogenesis and hydrolytic enzyme secretion in Candida albicans.

The effect of diallyl sulphide (DAS) and diallyl disulphide (DADS) on secretion of hydrolytic enzymes and dimorphism has been investigated in two strains of Candida albicans. Minimum inhibitory concentration (MIC) of DADS and DAS was determined to be 500 µg/ml and 40 µg/ml, respectively for a clinical isolate (accession #3043) and 450 µg/ml and 50 µg/ml, respectively, for a reference strain (ATCC 90028). At one-half of the minimum inhibitory concentration (MIC), DAS and DADS inhibited proteinase secretion by 24% and 35%, respectively, in the clinical strain, and 28% and 44%, respectively, in the reference strain. Inhibition of phospholipase secretion at one-half MIC of DAS and DADS was 27% and 60%, respectively, in the clinical strain and 31% and 64%, respectively, for the reference strain. Hyphal induction at 300 min in the reference strain was 15% (at one-half MIC of DAS) and 5% (at one-half the MIC of DADS) as compared to control (90% hyphal formation). Hyphal induction in the clinical strain was 16% (one-half the MIC of DAS) and 8% (one-half the MIC of DADS) compared to 95% in the control. To conclude, both DAS and DADS significantly inhibit proteinase, phospholipase secretion and dimorphism in C. albicans. These compounds can therefore be explored for their therapeutic potential against C. albicans.

Effect of diallyldisulphide on an antioxidant enzyme system in Candida species.

This study was carried out to show the effect of diallyldisulphide (DADS), an important organosulphur compound found in garlic (Allium sativum), on antioxidant systems in Candida species. Changes in antioxidant metabolites and antioxidant activity in the presence of DADS were found in Candida albicans and Candida tropicalis. Candida cells were treated with sublethal concentrations of DADS. DADS caused a decrease in the activity of all antioxidant enzymes except catalase, resulting in oxidative stress and damaged cells. The amount of oxidative stress generated by DADS was found to be a function of its concentration. A significant decrease in superoxide dismutase, glutathione-S-transferase, and glutathione peroxidase activities but an increase in catalase activity were observed. Increased levels of lipid peroxidation and decreased levels of glutathione were observed in treated cells. Activity of glucose-6-phosphate dehydrogenase decreased significantly following DADS treatment and could be correlated with a decrease in glutathione concentration in both Candida species. These results indicate that diallyl disulphide acts as a pro-oxidant to Candida species and hence may act as a potent antifungal in the management of candidiasis.

Proton translocating ATPase mediated fungicidal activity of eugenol and thymol.

Eugenol (1) and thymol (2) exhibit excellent fungicidal activity against pathogenic yeasts, including isolates resistant to azoles. The rapid irreversible action of compound-1 and compound 2 on fungal cells suggested a membrane-located target for their action. We investigated their effect on H(+)-ATPase mediated H(+)-pumping by various Candida species. Both compounds inhibit H(+)-ATPase activity at their respective MIC values--500 and 100 µg/ml. Glucose stimulated H(+)-extrusion was also inhibited significantly by compound 1 and compound 2. Inhibition of H(+)-ATPase leads to intracellular acidification and cell death. Inhibition of cell growth and H(+)-efflux by test compounds suggests that their antifungal properties are related to their inhibitory effects on H(+)-ATPase.

Antifungal activities of Ocimum sanctum essential oil and its lead molecules.

Aqueous extracts and oils of five Indian medicinal plants, traditionally used for their antimicrobial activities, were evaluated against two of the most prevalent Candida species causing candidiasis, C. albicans and C. tropicalis. Of these plant materials, three showed varying degrees of antifungal activity against both species. Tulsi (Ocimum sanctum Linn.) essential oil (TEO) was found to be the most effective, followed by Peppermint essential oil, and Aloe vera aqueous leaf extract. The product with the lowest MIC was further studied along with its lead molecules to explore the possible mechanism of action of the most active constituents. Eugenol, methyl eugenol, linalool, and 1, 8-cineole, along with TEO were then evaluated at the same. The pattern and extent of inhibition was studied using growth and WST1 cytotoxicity assays. Proton pumps are important for growth and metabolism of Candida species and so H+ extrusion studies were performed to explore the possible mechanism of the test compounds. Linalool was the most active constituent of TEO, whereas inhibition of H+ extrusion appeared to be a synergistic function of the lead molecules.