Anti-biofilm activity of a sophorolipid-amphotericin B niosomal formulation against Candida albicans

Sophorolipids (SLs) have gained interest in the pharmaceutical industries due to their anti-microbial, anti-adhesive and anti-biofilm properties. In the present study, the production of SL was increased by using low-cost media components. The potential of a SL-based niosomal formulation of amphotericin B (AmB) was determined against biofilm of the opportunistic fungal pathogen Candida albicans. In-house prepared SL-AmB niosomes were characterized by different microscopic techniques. The mean entrapment efficiency of AmB within SL-AmB niosome was 63.20% ± 3.86. The cytotoxicity of SL-AmB on mature C. albicans biofilm was compared with an expensive, marketed drug, viz. phosome (a liposomal formulation of AmB). Fewer hyphae were observed in C. albicans biofilm treated with SL-AmB niosome whereas more budding cells were found in phosome treated biofilm. The present study has established the affordable production of SL and the suitability of this approach for delivery of poorly soluble drugs such as AmB against candidiasis infections.     

Enhanced activity of antifungal drugs using natural phenolics against yeast strains of Candida and Cryptococcus

Aims: Determine whether certain, natural phenolic compounds enhance activity of commercial antifungal drugs against yeast strains of Candida and Cryptococcus neoformans. Methods and Results: Twelve natural phenolics were examined for fungicidal activity against nine reference strains of Candida and one of C. neoformans. Six compounds were selected for synergistic enhancement of antifungal drugs, amphotericin B (AMB), fluconazole (FLU) and itraconazole (ITR). Matrix assays of phenolic and drug combinations conducted against one reference strain, each, of Candida albicans and C. neoformans showed cinnamic and benzoic acids, thymol, and 2,3‐ and 2,5‐dihydroxybenzaldehydes (‐DBA) had synergistic interactions depending upon drug and yeast strain. 2,5‐DBA was synergistic with almost all drug and strain combinations. Thymol was synergistic with all drugs against Ca. albicans and with AMB in C. neoformans. Combinations of benzoic acid or thymol with ITR showed highest synergistic activity. Of 36 combinations of natural product and drug tested, none were antagonistic. Conclusions: Relatively nontoxic natural products can synergistically enhance antifungal drug activity, in vitro. Significance and Impact of the Study: This is a proof‐of‐concept, having clinical implications. Natural chemosensitizing agents could lower dosages needed for effective chemotherapy of invasive mycoses. Further studies against clinical yeast strains and use of animal models are warranted.     

Antifungal pharmacodynamic characteristics of amphotericin B against Trichosporon asahii, using time-kill methodology

We determined the MIC of amphotericin B against 45 Trichosporon asahii isolates from various clinical and environmental sources, and used in vitro time-kill methods to characterize the relationship between amphotericin B concentrations and MIC for four representative T. asahii isolates. Amphotericin B had concentration-dependent antifungal activity. MICs ranged from 0.5 to 16 microg/ml, and most T. asahii isolates (76%, 34/45) were inhibited at safely achievable amphotericin B serum concentrations (< or = 2 microg/ml). However, 40% (18/45) of isolates were not killed at these concentrations (MFCs from 1.0 to 32 microg/ml). At concentrations > or = 2 x MIC, amphotericin B exhibited fungicidal activity (< 99.9% reduction in CFU) over a 12-hr time-period; the maximal effect was achieved at > or =4 x MIC. Susceptibility testing confirmed the resistance of T. asahii to amphotericin B, and in vitro pharmacodynamic results also suggest that amphotericin B is not suitable therapy for T. asahii infection.     

Fungispecificity of fluconazole against Candida albicans

Candida albicans is an opportunistic pathogen of human mucosal surfaces. Colonization of oral and vaginal mucosa by this yeast is antagonized by the resident normal bacterial population. However, antibacterial therapy can alter the normal flora to allow fungal cells to attach, grow and invade host tissues. We studied the antimicrobic activity of fluconazole against clinical isolates of oral and vaginal bacteria and Candida albicans in vitro and in vivo by scanning and transmission electron microscopy; we also compared the bactericidal activity of fluconazole with clotrimazole in vitro by microbiologie assay. Fluconazole lysed fungi but did not change the ultrastructure of bacteria. Clotrimazole, but not fluconazole, was bactericidal against lactobacillus and streptococcus, the principal species of the oral and vaginal cavities. We conclude that Candida albicans, but not oral and vaginal bacteria, is susceptible to fluconazole. These observations help explain the antimycotic specificity of fluconazole and its efficacy against candidiasis in humans.     

Antifungal activities of origanum oil against Candida albicans

The antimicrobial properties of volatile aromatic oils from medicinal as well as other edible plants has been recognized since antiquity. Origanum oil, which is used as a food flavoring agent, possesses a broad spectrum of in vitro antimicrobial activities attributed to the high content of phenolic derivatives such as carvacrol and thymol. In the present study, antifungal properties of origanum oil were examined both in vitro and in vivo. Using Candida albicans in broth cultures and a micro dilution method, comparative efficacy of origanum oil, carvacrol, nystatin and amphotericin B were examined in vitro. Origanum oil at 0.25 mg/ml was found to completely inhibit the growth of C. albicans in culture. Growth inhibitions of 75% and >50% were observed at 0.125 mg/ml and 0.0625 mg/ml level, respectively. In addition, both the germination and the mycelial growth of C. albicans were found to be inhibited by origanum oil and carvacrol in a dose-dependent manner. Furthermore, the therapeutic efficacy of origanum oil was examined in an experimental murine systemic candidiasis model. Groups of mice (n = 6) infected with C. albicans (5 × LD₅₀) were fed varying amounts of origanum oil in a final vol. of 0.1 ml of olive oil (vehicle). The daily administration of 8.6 mg of origanum oil in 100 l of olive oil/kg body weight for 30 days resulted in 80% survivability, with no renal burden of C. albicans as opposed to the group of mice fed olive oil alone, who died within 10 days. Similar results were obtained with carvacrol. However, mice fed origanum oil exhibited cosmetically better clinical appearance compared to those cured with carvacrol. The results from our study encourage examination of the efficacy of origanum oil in other forms of systemic and superficial fungal infections and exploration of its broad spectrum effect against other pathogenic manifestations including malignancy.     

The synergistic antifungal activity of resveratrol with azoles against Candida albicans

Candida albicans is one of the most common clinical pathogenic microorganisms and it is becoming a serious health threat, particularly to immunocompromised populations. Drug resistance of Candida species has also frequently emerged, and combination therapy for fungal infections has attracted considerable attention. In this study, we established the Qinling Mountains myxobacterial secondary metabolites library and a synergic assay in combination with ketoconazole against C. albicans was introduced for metabolites screening. Two active compounds with synergic anticandidal activities were obtained, which were identified as trans-resveratrol and cis-resveratrol. According to our study, resveratrol can reduce the dosage to 1/64 of ketoconazole as well as itraconazole. Furthermore, synergistic anticandidal activity of resveratrol combined with azoles was verified against a panel of clinical C. albicans isolates, and the combination strategy enhanced the azoles susceptibility of three fluconazole-resistant isolates. These findings suggest that resveratrol enhances the efficacy of azoles and provides a promising application in therapy of C. albicans infection.     

Development of amphotericin B liposomes bearing antibody specific to Candida albicans

Summary Liposomes expressing external antibody specific for Candida albicans and encapsulating amphotericin B were developed and characterized in this study. Antibody was first modified by the covalent attachment of palmitic acid residues. Liposomes were produced by reverse-phase evaporation and modified antibody was incorporated into these liposomes via the hydrophobic interaction between the palmitic acid and the phospholipids composing the liposomes. The liposomes were characterized as to the amount of amphotericin B by spectroscopy and for the presence of antibody by protein analysis and secondary immunolabeling by fluorescent and electron microscopic methods. Immunogold labeling showed that the antibody was being expressed externally on the liposomes in the electron microscopic studies and the specificity of these liposomes for C. albicans was observed by secondary immunofluorescence.     

Evaluation of the antifungal photodynamic activity of Thymophylla pentachaeta extracts against Candida albicans and its virulence factors

BackgroundCandida albicans is one of the most common causative of opportunistic infections. Treatment of candidiasis is challenging considering the few antifungal drugs available and the increase in resistance. Antimicrobial photodynamic therapy (aPDT) is a recently developed therapeutic option that combines a non-toxic photosensitizer (PS) and light to kill the microbial pathogens. Targeting virulence, defined as the ability of a pathogen to cause overt disease, represents another attractive target for the development of novel antifungal agents. Thymophylla pentachaeta (DC.) Small var. belenidium (DC.) is an endemic plant from Argentina in which the presence of thiophenes, biologically active compounds whose antifungal activity is enhanced by irradiation with Ultraviolet A (UVA), have been already described.PurposeThe purpose of this study was to evaluate the photodynamic antifungal activity of hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and methanol (MeOH) extracts from T. pentachaeta var. belenidium and their inhibitory effects on C. albicans virulence factors as well as biofilm formation and eradication.Study Design/MethodsAntifungal photodynamic activity of Hex, DCM, EtOAc and MeOH extracts from different parts of the plant were assessed with the microbroth dilution, bioautography and the time-kill assays, under light and darkness conditions. The capacities of the most active extracts of inhibiting Candida virulence factors (adherence to epithelial cells, germ tube and pseudomycelium formation and hydrolytic enzyme secretion) were assessed. In addition, the activity against biofilm formation and eradication has been investigated by reaction with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) that quantifies living cells in these structures.ResultsHex and DCM extracts from T. pentachaeta roots exhibited high photodynamic antifungal activity against C. albicans [Minimal fungicide concentrations (MFCs)= 7.8 µg/ml] under UVA light irradiation. Chemical analysis of active extracts (Hex and DCM from roots) revealed the presence of photoactive thiophenes. Both extracts generate reactive oxygen species through type I and II mechanisms. These extracts, at sub-inhibitory concentrations, under light conditions decreased the adherence of C. albicans to Buccal Epithelial Cells (BEC), inhibited germ tube formation and reduced esterase production. Finally, they demonstrated activity against preformed biofilms submitted to irradiation (MFCs= 3.91 µg/ml and 15.63 µg/ml for Hex and DCM extracts, respectively).ConclusionTaking together, results demonstrated the strong photodynamic effects of T. pentachaeta root extracts under UVA irradiation, making them valuable alternatives to the already established antifungal drugs against C. albicans.     

土槿乙酸对白假丝酵母菌生物膜的抑制作用

目的 探讨土槿乙酸（pseudolaric acid B,PAB）对体外白假丝酵母菌生物膜的影响。方法 甲基四氮盐（XTT）法检测不同浓度PAB和AMB（两性霉素B）对白假丝酵母菌生物膜的抑制作用。血清芽管试验检测不同浓度PAB对芽管生成的影响。结果 PAB对白假丝酵母菌生物膜的SMIC50（抑制生物膜50%的药物浓度）为256~512 μg/mL;1024和512 μg/mL PAB对早期2 h生物膜的抑制率分别为（99.5±0.28）%和（97.1±0.38）%;512 μg/mL PAB对早期（2 h）、中期（8 h）及成熟期（24 h）生物膜的抑制率分别为（97.1±0.38）%、（90.4±0.32）%和（80.1±0.67）%;不同浓度PAB的血清芽管试验显示,64 μg/mL PAB可以完全抑制白假丝酵母菌的出芽生长,16 μg/mL PAB可以抑制83.5%的白假丝酵母菌出芽生长。结论 PAB对体外白假丝酵母菌生物膜有抑制作用,对白假丝酵母菌的出芽生长过程抑制作用显著。     

Enhancement of Amphotericin B Activity Against Candida albicans by Superoxide Radical

This study aimed to examine the involvement of oxidative damage in amphotericin B (AmB) activity against Candida albicans using the superoxide (O2-) generator paraquat (PQ). The effects of PQ on AmB activities against growth, viability, membrane permeability and respiration were examined in a wild-type parent strain (K) and a respiration-deficient mutant (KRD-19) since PQ-induced superoxide generation depends on respiration. In the parent strain, the minimal inhibitory concentration (MIC) of AmB, 0.25 microg/ml, tested with a liquid culture was lowered to 0.025 microg/ml by 1 mM PQ. Such a PQ-induced decrease in the MIC value of AmB was minimal in the mutant. Similar PQ-induced enhancement of AmB activity toward the parent strain was also observed with growth on an agar medium. In viability tests, when candidal cells were exposed to AmB (0.1 microg/ml) for I h, the lethality of AmB was enhanced by 1 mM PQ only in the parent strain. Exogenous superoxide dismutase and catalase failed to diminish the enhancing effect of PQ on the growth inhibitory activity of AmB in the parent strain, suggesting an interaction between superoxide and AmB in candidal cells. The enhancement of AmB activity by PQ, observed preferentially in the wild-type strain, can be explained by extensive superoxide generation depending on respiration. These results suggest that oxidative damage induced by superoxide is involved in AmB activity against C. albicans.     

Silver colloidal nanoparticles: antifungal effect against adhered cells and biofilms of Candida albicans and Candida glabrata

The aim of this study was to evaluate the effect of silver nanoparticles (SN) against Candida albicans and Candida glabrata adhered cells and biofilms. SN (average diameter 5 nm) were synthesized by silver nitrate reduction with sodium citrate and stabilized with ammonia. Minimal inhibitory concentration (MIC) tests were performed for C. albicans (n = 2) and C. glabrata (n = 2) grown in suspension following the Clinical Laboratory Standards Institute microbroth dilution method. SN were applied to adhered cells (2 h) or biofilms (48 h) and after 24 h of contact their effect was assessed by enumeration of colony forming units (CFUs) and quantification of total biomass (by crystal violet staining). The MIC results showed that SN were fungicidal against all strains tested at very low concentrations (0.4–3.3 μg ml^?1). Furthermore, SN were more effective in reducing biofilm biomass when applied to adhered cells (2 h) than to pre-formed biofilms (48 h), with the exception of C. glabrata ATCC, which in both cases showed a reduction ～90%. Regarding cell viability, SN were highly effective on adhered C. glabrata and respective biofilms. On C. albicans the effect was not so evident but there was also a reduction in the number of viable biofilm cells. In summary, SN may have the potential to be an effective alternative to conventional antifungal agents for future therapies in Candida-associated denture stomatitis.     

米卡芬净对分离自中国的念珠菌和曲霉临床株体外抑菌活性的研究

目的了解新型抗真菌药物米卡芬净(micafungin,MFG)对分离自中国的念珠菌和曲霉临床株的体外抑菌活性。方法参照CLSI(Clinical and Laboratory Standards Institute,以前为NCCLS)制定的M27-A2和M38-A方案测定86株念珠菌和35株曲霉的最低抑菌浓度(MIC)或最低有效浓度(MEC)。结果MFG对大多数念珠菌属和曲霉属均有较好的抑菌作用。对念珠菌属的MIC90从高到低依次为:氟康唑(FLC)敏感的白念珠菌、热带念珠菌、光滑念珠菌为0.125μg/ml,FLC耐药和剂量依赖敏感株为0.25μg/ml,克柔念珠菌为0.5μg/ml,近平滑念珠菌8μg/ml,季也蒙念珠菌>16μg/ml。MFG对烟曲霉的MEC90为≤0.03μg/ml,对非烟曲霉的曲霉属MEC90为0.06μg/ml。MFG与唑类药物、两性霉素B(AMB)不存在交叉耐药,对FLC耐药的念珠菌、伊曲康唑耐药的曲霉、AMB不敏感的曲霉均有好的抑菌活性。结论MFG对多数念珠菌属和曲霉属(包括对唑类耐药和AMB不敏感的菌株)有较好的体外抑菌作用。     

Silver nanoparticles: influence of stabilizing agent and diameter on antifungal activity against Candida albicans and Candida glabrata biofilms

Aim: The purpose of this work was to evaluate the size‐dependent antifungal activity of different silver nanoparticles (SN) colloidal suspensions against Candida albicans and Candida glabrata mature biofilms. Methods and Results: The research presented herein used SN of three different average sizes (5, 10 and 60 nm), which were synthesized by the reduction of silver nitrate through sodium citrate and which were stabilized with ammonia or polyvinylpyrrolidone. Minimal inhibitory concentration (MIC) assays were performed using the microdilution methodology. The antibiofilm activity of SN was determined by total biomass quantification (by crystal violet staining) and colony forming units enumeration. MIC results showed that all SN colloidal suspensions were fungicidal against the tested strains at very low concentrations (0·4–3·3 μg ml^?1). With regard to biomass quantification, SN colloidal suspensions were very effective only against C. glabrata biofilms, achieving biomass reductions around 90% at a silver concentration of 108 μg ml^?1. In general, all SN suspensions promoted significant log₁₀ reduction of the mean number of cultivable biofilm cells after exposure to silver concentrations at or higher than 108 μg ml^?1. Moreover, the results showed that the particle size and the type of stabilizing agent used did not interfere in the antifungal activity of SN against Candida biofilms. Conclusions: This study suggests that SN have antifungal therapeutic potential, but further studies are still required namely regarding formulation and delivery means. Significance and Impact of the Study: SN may contribute to the development of new strategies for the improvement of oral health and quality of life particularly of the complete denture wearers.     

Effect of antifungal gels incorporated into a tissue conditioning material on the growth of Candida albicans

doi:10.1111/j.1741‐2358.2009.00337.x
Effect of antifungal gels incorporated into a tissue conditioning material on the growth of Candida albicans Objective: The aim of this study was to examine the effectiveness of antifungal gels incorporated into a tissue conditioner which inhibits the growth of Candida albicans in vitro. Background: The release of drugs from relining materials has been demonstrated earlier. However, the incorporation of antifungal agents in gel form has not yet been studied. Materials and methods: Visco‐gel^® tissue conditioner was prepared with chlorhexidine digluconate and miconazole in gel form in a concentration of 5, 10, 15, 20 and 25% by volume. Sample discs were prepared and placed on Sabouraud Dextrose Agar (SDA) plates which had been previously inoculated with C. albicans, and incubated aerobically at 37°C. To investigate antifungal activity over time, Visco‐gel discs containing 20%v/v miconazole were prepared and immersed in water for different time periods before being placed on SDA plates inoculated with C. albicans. Results: Chlorhexidine digluconate gel added to tissue conditioner had no inhibition effect on the growth of C. albicans. Incorporation of miconazole gave a dose‐related inhibitory effect on candidal growth. Immersion of the discs in water showed an inverse relationship between time of immersion and degree of inhibition. Conclusion: Miconazole added in gel form to Visco‐gel^® had an inhibitory effect on the growth of C. albicans in vitro.     

High-dose methylprednisolone influences the physiology and virulence of Candida albicans ambiguously and enhances the candidacidal activity of the polyene antibiotic amphotericin B and the superoxide-generating agent menadione

Although exposure of Candida albicans cells to high-dose (4 mM) methylprednisolone stimulated microbial growth, germination rate in serum and phospholipase release, it also promoted the recognition of C. albicans cells by polymorphonuclear leukocytes. Pretreatment of C. albicans cells with methylprednisolone did not result in any increase in the pathogenicity of the fungus in intraperitoneal and intravenous mouse assays. Therefore, the virulence of C. albicans is unlikely to increase in patients treated with comparably high-dose methylprednisolone on skin and mucosal membranes. Methylprednisolone treatments also increased the production of conjugated dienes and thiobarbituric acid-reactive substances, and the menadione sensitivity of C. albicans cells, which can be explained by a significant decrease in the specific activities of several antioxidant enzymes. The combination of methylprednisolone with oxidants, e.g. in topical applications, may be of clinical importance when the predisposition to candidiasis is high. Methylprednisolone treatments negatively affected membrane fluidity and decreased the antifungal effects of both the polyene antibiotic nystatin and the ergosterol biosynthesis inhibitor lovastatin, and also enhanced the deleterious effects of the polyene antimycotic amphotericin B on C. albicans cells. These corticosteroid-polyene drug interactions should be considered in the treatment of C. albicans infections in patients with prolonged topical application of corticosteroids.