Detection of Candida albicans in disseminated candidosis by immunofluorescence staining

An indirect immunofluorescence (IF) method using rabbit anti-Candida albicans was used to detect C. albicans in blood samples of 12 patients with systemic candidosis defined clinically, histologically and by blood cultures. Positive staining of C. albicans could be detected in all of the patients. The findings suggest that IF-method offers a more rapid method in the diagnosis of disseminated candidosis.     

Antifungal activity and mode of action of silver nano-particles on Candida albicans

In this study, the antifungal effects of silver nano-particles (nano-Ag) and their mode of action were investigated. Nano-Ag showed antifungal effects on fungi tested with low hemolytic effects against human erythrocytes. To elucidate the antifungal mode of action of nano-Ag, flow cytometry analysis, a glucose-release test, transmission electron microscopy (TEM) and the change in membrane dynamics using 1,6-diphenyl-1,3,5-hexatriene (DPH), as a plasma membrane probe, were performed with Candida albicans. The results suggest nano-Ag may exert an antifungal activity by disrupting the structure of the cell membrane and inhibiting the normal budding process due to the destruction of the membrane integrity. The present study indicates nano-Ag has considerable antifungal activity, deserving further investigation for clinical applications. K.-J. Kim and W. S. Sung contributed equally to this work and should be considered co-first authors.     

Factors involved in the adherence of Candida albicans and Candida tropicalis to protein-adsorbed surfaces

The adherence of Candida albicans and C. tropicalis to protein-adsorbed surfaces was investigated with surface-modified glass slides to which serum or salivary proteins were covalently bound. A specific adherence like a ligand-receptor interaction was observed between C. albicans and mucin- or salivary protein-immobilized glass slides. This interaction was eliminated by deglycosylation of the slides, suggesting that the receptor may be an oligosaccharide(s) contained mucin or saliva. A similar specific interaction was also observed between C. tropicalis and fibrinogen-immobilized glass surfaces. When the numbers of adherent cells to deglycosylated protein-immobilized glass glides were plotted against zeta potentials and contact angles of these protein-immobilized glass slides, a significant correaltion was observed between the numbers of adherent cells and zeta potentials in the case of C. albicans (r = –0.87), whereas a significant correlation was observed between cell numbers and contact angles (r = 0.82) in the case of C. tropicalis. These results suggest that the forces governing the adherence of fungi to pellicle in dentures may vary depending upon the surface properties of fungi and substrate.     

Cell-associated collagenolytic activity by Candida albicans

Cell associated collagenolytic activity of Candida albicans was quantified by measuring the degradation of synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which is a specific substrate for collagenase, by the freeze-thaw procedure method. This collagenolytic activity was enhanced by cells cultured in the presence of bovine serum albumin (BSA) in culture medium. However, this activity was inhibited in the presence of ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), but not by the serine proteinase inhibitor p-amidinophenyl methanesulfonyl fluoride (APMSF), nor the aspartyl proteinase inhibitor pepstatin A. These results suggested the presence of a metalloenzyme on pericellular C. albicans. This revised version was published online in June 2006 with corrections to the Cover Date.     

beta-Galactosidase of Kluyveromyces lactis (Lac4p) as reporter of gene expression in Candida albicans and C. tropicalis.

Summary Vectors containing fusions of the Candida albicans ACT promoter to heterologous genes were constructed and transformed into a C. albicans host strain. -Galactosidase (Lac4p) activity was detected in transformants carrying an ACT fusion to the Kluyveromyces lactis LAC4 gene, while fusions to the Escherichia coli lacZ gene and to other heterologous genes were not expressed. Lac4p was also produced by C. tropicalis transformants carrying the ACT/LAC4 fusion. Plasmids in transformed C. albicans strains were present either as free multimers in high copy number or, more frequently, integrated into the genome in low copy number yielding high and low LAC4 mRNA and Lac4p expression levels, respectively. Lac4p-expressing transformants of C. tropicalis, but not of C. albicans, were able to utilize lactose as sole carbon source. An ACT/LAC4 fusion was not differentially expressed during the yeast and hyphal growth phases of C. albicans, indicating that the ACT promoter is not regulated during morphogenesis. These results define the first reporter gene system for convenient monitoring of gene expression in Candida species.     

Anti-Candida and mode of action of two newly synthesized polymers: a modified poly (methylmethacrylate-co-vinylbenzoylchloride) and a modified linear poly (chloroethylvinylether-co-vinylbenzoylchloride) with special reference to Candida albicans and Candida tropicalis

Polymeric antimicrobial agents represent a new and important direction that is developing in the field of antimicrobial agents. Antimicrobial activity of two newly synthesized polymers: a modified poly (methylmethacrylate-co-vinylbenzoylchloride) and a modified linear poly (chloroethylvinylether-co-vinylbenzoylchloride) have been investigated and found to be active. Both polymers have showed a broad antimicrobial activity against C. albicans and C. tropicalis. Minimal inhibitory concentrations (MIC's) for poly (methylmethacrylate-co-vinylbenzoyl chloride) were 100, 75 and 100 microg/ml in case of C. albicans (ATCC 2091), C. albicans (SC5314) and C. tropicalis, respectively. However, polycholoroethylvinylether-covinylbenzoylchloride inhibited C. albicans (ATCC 2091), C. albicans (SC5314) and C. tropicalis with minimum inhibitory concentration values (MIC's) of 150 microg/ml against the three tested Candida strains. Mode of action studies of both polymers on the medically important yeasts, C. albicans and C. tropicalis revealed that poly (methylmethacrylate-co-vinylbenzoylchloride) induced cytotoxicity, DNA damage, and altered cell permeability and morphology, which was manifested as aggregated and swollen yeast cells (C. albicans ATCC 2091) by fluorescent microscopy examination. Poly (chloroethylvinylether-co-vinylbenzoylchloride) increased cell permeability, and respiration for C. albicans and C. tropicalis. The tested polymers at 50 microg/ml had pronounced effects on C. albicans and C. tropicalis cell wall phosphopeptidomannane, proteins, sugars and phosphorus. Generally, the two polymers proved effective against the tested microorganisms, but growth inhibitory effect varied according to the composition of the polymer active group. Many investigators consider polymeric antimicrobial agents as a potential new approach for enhancing the efficiency of some existing antimicrobial agents, including prolonged activity, reduce their toxicity, as well as reduce the environmental issues associated with product use.     

A novel antifungal from an Actinomadurae with preferential activity against the mycelial phase ofCandida albicans

Summary Sch 40873, a novel antifungal compound isolated from the fermentation broth of anActinomadura spp. was discovered in an assay designed to detect compounds with preferential activity against the invasive mycelial form ofCandida albicans. The geometric mean MIC of Sch 40873 against sevenCandida spp. in Sabouraud dextrose broth (yeast phase) was 58 g/ml and in Eagles minimum essential medium (mycelial phase) was <0.03 g/ml. Sch 40873 demonstrated slight in vivo topical activity in a hamster vaginal model.     

Effect of Candida albicans dsDNA in Gastrointestinal Candida Infection

Neonates are highly sensitive to infections because they are biased to develop Th2 immune responses. When exposed to certain agents, such as DNA vaccines or CpG DNA motifs, neonates are capable to mount adult-like Th1 protective responses. This study investigates the capacity of Candida albicans (C. albicans) dsDNA to induce host resistance in newborn mice against gastrointestinal C. albicans infection. The protective properties of dsDNA are related to an increased number of spleen CD4+ T cells secreting IFN-γ. In infected DNA-treated mice, an enhanced production of IFN-γ by Peyer’s patch cells was observed together with reduced colonization and histopathological changes in the stomach. Our results indicated that C. albicans dsDNA administration in neonates elicited the protective immune response against gastrointestinal Candida infection.     

The impact of polyene,azole, and DNA analogue antimycotics on the cell surface hydrophobicity of Candida albicans and Candida tropicalis in HIV infection

Oral candidiasis is the most common opportunistic infection in individuals infected with the human immunodeficiency virus. Though Candida albicans is the major aetiological agent, non-albicans species such Candida tropicalis are now emerging as important agents of such infection. The Candida cell surface hydrophobicity (CSH) is considered a critical factor contributing to its colonization potential and virulence. It is also known that brief exposure to sub-cidal concentrations of antifungal agents is a likely scenario in the oral environment where the administered drugs are diluted continuously due to the flushing action of saliva. Hence the objective of the present study was to compare the CSH of 10 isolates each of C. albicans and C. tropicalis from HIV-infected individuals following brief exposure (1hour) of isolates to sub-therapeutic concentrations of nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine. The CSH was assessed by a previously described biphasic aqueous-hydrocarbon assay. The mean percentage reduction of CSH of C. albicans following brief exposure to nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine was27.33 (p < 0.001), 21.34 (p < 0.05), 11.74 (p > 0.05), 18.4 (p > 0.05) and 14.64 (p > 0.05) respectively. The mean percentage reduction of CSH of C. tropicalis following brief exposure to nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine was 33.81 (p < 0.01), 28.88 (p < 0.01), 12.6 (p > 0.05), 21.53(p > 0.05) and 17.68 (p > 0.05) respectively. A significant inter-species variation in CSH was observed for nystatin and amphoterecin B. Overall the results reveal that the CSH of C. albicans is affected to a significantly lesser degree compared with C. tropicalis when exposed to the antifungals. These data further illustrate another mode of action of antifungals on Candida leading to a reduction in the CSH and thereby the yeast adherence to host tissues. This revised version was published online in June 2006 with corrections to the Cover Date.     

A simple PCR/RFLP analysis can differentiate between Candida albicans, Aspergillus niger, and Aspergillus fumigatus

The clinical management of immunocompromised patients depends on the rapid identification of infectious agents such as fungal pathogens. The procedure described here for accomplishing this uses a sensitive polymerase chain reaction method, previously reported, combined with restriction-enzyme digestion to distinguish between Candida and Aspergillus species and to classify Aspergillus strains.     

Molecular Analysis and Susceptibility Profiling of Candida albicans Isolates from Immunocompromised Patients in South India

The genetic diversity and in vitro antifungal susceptibility profiles of 55 Candida albicans from immunocompromised patients were studied. PCR based analysis of the transposable intron in the 25S rDNA revealed 39 genotype A, 4 genotype B and 12 genotype C isolates. Serotype analysis categorized 52 isolates as serotype A and 3 as serotype B. All strains were susceptible to micafungin, 5-flucytosine and miconazole, whereas resistance against amphotericin B (3.6%), fluconazole (3.6%), itraconazole (7.3%) and voriconazole (5.5%) was observed. No association was seen between antifungal resistance and genotype/serotype status.     

Pseudomonas aeruginosa, Candida albicans, and device-related nosocomial infections: implications, trends, and potential approaches for control

For many years, device-associated infections and particularly device-associated nosocomial infections have been of considerable concern. Recently, this concern was heightened as a result of increased antibiotic resistance among the common causal agents of nosocomial infections, the appearance of new strains which are intrinsically resistant to the antibiotics of choice, and the emerging understanding of the role biofilms may play in device-associated infections and the development of increased antibiotic resistance. Pseudomonas aeruginosa and Candida albicans are consistently identified as some of the more important agents of nosocomial infections. In light of the recent information regarding device-associated nosocomial infections, understanding the nature of P. aeruginosa and C. albicans infections is increasingly important. These two microorganisms demonstrate: (1) an ability to form biofilms on the majority of devices employed currently, (2) increased resistance/tolerance to antibiotics when associated with biofilms, (3) documented infections noted for virtually all indwelling devices, (4) opportunistic pathogenicity, and (5) persistence in the hospital environment. To these five demonstrated characteristics, two additional areas of interest are emerging: (a) the as yet unclear relationship of these two microorganisms to those species of highly resistant Pseudomonas spp and Candida spp that are of increasing concern with device-related infections, and (b) the recent research showing the dynamic interaction of P. aeruginosa and C. albicans in patients with cystic fibrosis. An understanding of these two opportunistic pathogens in the context of their ecosystems/biofilms also has significant potential for the development of novel and effective approaches for the control and treatment of device-associated infections.     

Specificity of drug transport mediated by CaMDR1: a major facilitator of Candida albicans

CaMDR1 encodes a major facilitator superfamily (MFS) protein inCandida albicans whose expression has been linked to azole resistance and which is frequently encountered in this human pathogenic yeast. In this report we have overexpressed CaMdr1p inSf9 insect cells and demonstrated for the first time that it can mediate methotrexate (MTX) and fluconazole (FLC) transport. MTX appeared to be a better substrate for CaMdr1p among these two tested drugs. Due to severe toxicity of these drugs to insect cells, further characterization of CaMdr1p as a drug transporter could not be done with this system. Therefore, as an alternative, CaMdr1p and Cdr1p, which is an ABC protein (ATP binding cassette) also involved in azole resistance inC. albicans, were independently expressed in a common hypersensitive host JG436 ofSaccharomyces cerevisiae. This allowed a better comparison between the functionality of the two export pumps. We observed that while both FLC and MTX are effluxed by CaMdr1p, MTX appeared to be a poor substrate for Cdr1p. JG436 cells expressing Cdr1p thus conferred resistance to other antifungal drugs but remained hypersensitive to MTX. Since MTX is preferentially transported by CaMdr1p, it can be used for studying the function of this MFS protein.     

Requirement for nonprotonated drug molecules in the direct lethal action of miconazole against Candida albicans

At 10^–5 M, miconazole (MCZ) can exert a direct physicochemical cell-damaging lethal action against logarithmic phase yeasts of Candida albicans. The imidazole moiety of MCZ has a pKa 6.5. Thus, in media of pH >6.5 most drug molecules are nonprotonated (MCZ). Conversely, at pH < 6.5 the majority are protonated and carry a positive charge (MCZH⁺). Our earlier work suggesting that MCZ is required for direct lethal action was tested further. In support of such a requirement, we established a minimal lethal concentration of MCZ (i.e. 5×10^–6 M) that was relatively independent of pH, MCZ concentration, and MCZMCZH⁺ ratio.     

Molecular cloning and biochemical characterization of Candida albicans acyl-CoA:sterol acyltransferase, a potential target of antifungal agents

To determine whether Candida albicans acyl CoA:sterol acyltransferase (ASAT) can be a potential target enzyme for the protoberberine derivative (HWY-289), we have isolated a gene encoding Ca-ASAT and examined inhibitory effects of HWY-289 on the overexpressed Ca-ASAT. HWY-289 specifically inhibits Ca-ASAT in a non-competitive manner in vitro (IC(50) [9.2microM], K(i) [5.15microM]). The cloned CaARE2 gene (1830 nucleotides [nt]) encodes active Ca-ASAT protein that exhibits a calculated molecular mass of 71.3kDa. The amino acid sequence of CaAre2p is 33.4% and 35.1% identical to those of Saccharomyces cerevisiae ScAre1p and ScAre2p homologues, respectively. Recombinant and endogenous Ca-ASAT displayed identical patterns of inhibition upon exposure to HWY-289 and a preference for cholesterol and oleoyl-CoA as substrates. Northern blot analysis showed that CaARE2 was activated by HWY-289, but not by CI-976 (a human acyl-coenzyme A:cholesterol acyltransferase inhibitor), in a dose-dependent manner (up to 5mg/L), suggesting different selectivities of action between HWY-289 and CI-976 on Ca-ASAT activity.     

Induction of peroxisome proliferation and increase of catalase activity in yeast,Candida albicans,by cadmium

The effects of cadmium on the growth rate, catalase activity, and peroxisome proliferation in yeast,Candida albicans, were evaluated. The yeast growth was markedly inhibited by 1 mM cadmium at the initial hours. The toxic effect of cadmium on the cell growth persisted. The catalase activity of the cells treated with 1 mM Cd²⁺ first decreased, and then rose at 24 h to about 2.6 times that of the controls. The average number of peroxisomes per cell in the yeast treated with 1 mM Cd²⁺ was about sixfold higher than the control groups. The proliferation of peroxisomes and the increase of catalase activity following cadmium toxicity gives credence to the hypothesis that cadmium toxicity is related to its potential to induce oxidative stress in cells.     

Candida albicans strain-dependent modulation of pro-inflammatory cytokine release by in vitro oral and vaginal mucosal models

Candida albicans is a commensal organism at several sites and is a versatile, opportunistic pathogen. The underlying factors of pathogen and host associated with commensalism and pathogenicity in C. albicans are complex and their importance is largely unknown. We aimed to study the responses of oral epithelial (OEM) and vaginal epithelial models (VEM) to infection by oral and vaginal C. albicans strains to obtain evidence of inter-strain differences in pathogenicity and of site-specificity. Following inoculation of models, proinflammatory cytokines IL-1α, IL-1β, IL-6, IL-8 and prostaglandin E2 (PGE2) release were monitored and histological staining undertaken. Striking differences in strain behaviour and epithelial responses were observed. IL-1α, IL-1β and IL-8 release were significantly increased from the OEM in response to denture stomatitis strain NCYC 1467. Increased IL-8 release also followed infection of the OEM with both vaginal strains. Overall the VEM was relatively unresponsive to infection with either oral or vaginal strains under these conditions. Adherence and hyphal development were observed for all strains on both models although extensive, uniform tissue penetration was seen only with stomatitis strain NCYC 1467 on the OEM. Candidal strains were assayed for phospholipase (PL) and secreted aspartyl proteinase (SAP) activities where phospholipase (PL) activity was highest for strain NCYC 1467 although highest SAP activity was observed for vaginal strain NCPF 8112 in this assay. This is the first study to concurrently investigate cytokine production from oral and epithelial models using candidal strains originating from these respective mucosal sites from healthy and disease states. These data demonstrate significant differences in inflammatory responses of host epithelia to individual C. albicans strains.     

Distribution and phospholipase activity of Candida species in different denture stomatitis types

The aim of this study was to evaluate the correlation between frequency and phospholipase activity of Candida species and denture stomatitis according to Newton’s classification. Seventy-five complete denture wearers were evaluated for the presence of yeasts on the palatal mucosa by culture method. In addition, the number of yeast isolates producing phospholipase and amount of this enzyme were determined using egg yolk agar plate method. According to Newton’s classification, 25 denture wearers were with healthy palatal mucosa while 50 were with any types of denture stomatitis. The frequency of yeasts was linked to whether subjects had Type II or Type III, but not Type I denture stomatitis. Candida albicans was the most frequently isolated species in denture wearers with and without clinical signs of denture stomatitis and it was the only species produced phospholipase. Although the amount of phospholipase produced by the C. albicans isolates from denture wearers in control and Type II and III DS groups was not significantly different, there was statistically significant difference in the number of C. albicans isolates producing phospholipase between patients with and without clinical signs of DS.     

New antifungal agents that inhibit the growth of Candida Species: Dichlorinated 8-quinolinols

Five dichlorinated 8-quinolinols (2,5- 5,6-, 3,5-, 3,7-, and 4,5-dichloro-8-quinolinol) were tested against Candida albicans and C. Tropicalis in Sabouraud dextrosebroth with and without bovine serum. The 5,6-, 3,5-, and 3,7-dichloro-8-quinolinols proved to be more effective than the control, 5-fluorocytosine. In cytotoxicity tests employing baby hamster kidney (BHK) cells, all test agents proved to be more cytotoxic than the control. However, the minimum inhibitory concentration (MIC) of 3,5-dichloro-8-quinolinol to both fungi was only one tenth the cytotoxic dose,suggesting that the compound may be useful as a topical or systemic antifungal agent.This revised version was published online in October 2005 with corrections to the Cover Date.     

RTA2, a novel gene involved in azole resistance in Candida albicans

Widespread and repeated use of azoles, particularly fluconazole, has led to the rapid development of azole resistance in Candida albicans. Overexpression of CDR1, CDR2, and CaMDR1 has been reported contributing to azole resistance in C. albicans. In this study, hyper-resistant C. albicans mutant, with the above three genes deleted, was obtained by exposure to fluconazole and fluphenezine for 28 passages. Thirty-five differentially expressed genes were identified in the hyper-resistant mutant by microarray analysis; among the 13 up-regulated genes, we successfully constructed the rta2 and ipf14030 null mutants in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1. Using spot dilution assay, we demonstrated that the disruption of RTA2 increased the susceptibility of C. albicans to azoles while the disruption of IPF14030 did not influence the sensitivity of C. albicans to azoles. Meanwhile, we found that ectopic overexpression of RTA2 in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1 conferred resistance to azoles. RTA2 expression was found elevated in clinical azole-resistant isolates of C. albicans. In conclusion, our findings suggest that RTA2 is involved in the development of azole resistance in C. albicans.