Molecular subtyping of clinical isolates of <Emphasis Type="Italic">Candida albicans</Emphasis> and identification of <Emphasis Type="Italic">Candida dubliniensis</Emphasis>in Malaysia

The genotypes of 221 recent isolates of Candida albicans from various clinical specimens of 213 patients admitted to the University Malaya Medical Centre, Malaysia was determined based on the amplification of a transposable intron region in the 25 S rRNA gene. The analyses of 178 C. albicansisolated from nonsterile clinical specimens showed that they could be classified into three genotypes: genotype A (138 isolates), genotype B (38 isolates) and genotype C (2 isolates). The genotyping of 43 clinical isolates from sterile specimens showed that they belonged to genotype A (29 isolates), genotype B (10 isolates), genotype C (2 isolates) and genotype D (2 isolates). The overall distribution of C. albicans genotypes in sterile and nonsterile specimens appeared similar, with genotype A being the most predominant type. This study reported the identification of C. dubliniensis (genotype D) in 2 HIV-negative patients with systemic candidiasis, which were missed by the routine mycological procedure. The study demonstrated the genetic diversity of clinical isolates of C. albicans in Malaysia.     

Characterization of Switch Phenotypes in <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilms

The aim of this study was to characterize switch phenotypes in Candida albicans biofilms. Cells of Candida albicans 192887g biofilms (24 h) were resuspended and these together with their planktonic counterparts were separately inoculated on Lee’s medium agar supplemented with arginine and zinc, at 25 °C for 9 days, for colony formation. The different switch phenotypes, as reflected by varying colony morphologies, were then examined for their (i) stability under various growth conditions, (ii) carbohydrate assimilation profiles, (iii) susceptibility to the polyene antifungal, nystatin, (iv) adhering and biofilm-forming ability, (v) filamentation, and (vi) growth rate in yeast nitrogen base medium supplemented with 100 mM glucose. Our data showed that the frequency of phenotypic switching in C. albicans biofilms was approximately 1%. Compared with the planktonic yeasts, cells derived from candidal biofilms generated one of the phenotypes less frequently (Chi-square-tests: P = 0.017). The five phenotypes derived from the biofilm growth demonstrated differing profiles for carbohydrate assimilation, adhesion, biofilm formation, filamentation, and growth rate. These findings reported here, for the first time, imply that phenotypic switching in the candidal biofilms differs from that in the planktonic growth, and affects multiple biological attributes.     

Biofilm formation by <Emphasis Type="Italic">Candida albicans</Emphasis> isolated from intrauterine devices

Our survey revealed that infected intrauterine devices (IUDs) recovered from patients suffering from reproductive tract infections (RTIs) were tainted with Candida biofilm composed of a single or multiple species. Scanning electron microscopy (SEM) analysis of C. albicans biofilm topography showed that it consists of a dense network of mono- or multilayer of cells embedded within the matrix of extracellular polymeric substances (EPS). Confocal scanning laser microscopy (CSLM) and atomic force microscopy (AFM) images depicted that C. albicans biofilms have a highly heterogeneous architecture composed of cellular and noncellular elements with EPS distributed in the cell-surface periphery or at cell-cell interface. Biochemical analysis showed that EPS produced by C. albicans biofilm contained significantly reduced total carbohydrate (40%), protein (5%) and enhanced amount of hexosamine (4%) in contrast to its planktonic counterparts. The in vitro activity of antifungal agents amphotericin B, nystatin, fluconazole and chlorhexidine against pre-formed C. albicans biofilm, assessed using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay revealed increased resistance of these infectious biofilm (50% reduction in metabolic activity at a concentration of 8, 16, 64, 128 μg/ml respectively) in comparison to its planktonic form.     

The white-phase-specific gene<Emphasis Type="Italic"> WH11</Emphasis> is not required for white-opaque switching in<Emphasis Type="Italic"> Candida albicans</Emphasis>

Phenotypic switching between white and opaque cells is important for adaptation to different host environments and for mating in the opportunistic fungal pathogen Candida albicans. Genes that are specifically activated in one of the two cell types are likely to be important for their phenotypic characteristics. The WH11 gene is a white-phase-specific gene that has been suggested to be involved in the maintenance of the white-phase phenotype. To elucidate the role of WH11 in white-opaque switching, we constructed mutants of the C. albicans strain WO-1 in which the WH11 gene was deleted. The wh11 mutants were still able to form both white and opaque cells whose cellular and colony phenotypes were indistinguishable from those of the wild type. Deletion of WH11 also did not affect the activation and deactivation of the white-phase-specific WH11 promoter and the opaque-phase-specific OP4 and SAP1 promoters in the appropriate cell type. Finally, switching from the white to the opaque phase and vice versa occurred with the same frequency in wild-type and wh11 mutants. Therefore, the WH11 gene is not required for phenotypic switching, and its protein product seems to have other roles in white cells, which are dispensable after the switch to the opaque phase.Communicated by E. Cerdá-Olmedo     

Biodegradation of phenol and <Emphasis Type="Italic">m</Emphasis>-cresol by <Emphasis Type="Italic">Candida albicans</Emphasis> PDY-07 under anaerobic condition

Strain Candida albicans PDY-07 was used to study the anaerobic biodegradation of phenol and m-cresol as single and dual substrates in batch cultures. The strain had a higher potential to degrade phenol than m-cresol. The cell growth kinetics of batch cultures with various initial m-cresol concentrations was investigated, and the Haldane kinetic model adequately described the dynamic behavior of cell growth on m-cresol. When cells grew on the mixture of phenol and m-cresol, substrate interactions were observed. Phenol inhibited the utilization of m-cresol; on the other hand, m-cresol also inhibited the degradation of phenol. However, the presence of low-concentration phenol enhanced m-cresol biodegradation; 100 mg/l m-cresol could be completely degraded within a shorter period of time than m-cresol alone in the presence of 150–300 mg/l phenol. The maximum m-cresol biodegradation rate was obtained at the existence of 200 mg/l phenol. Phenol was preferably utilized by the strain as a carbon and energy source. In addition, a sum kinetics model was used to describe the cell growth behavior in binary mixture of phenol and m-cresol, and the interaction parameters were determined. The model adequately predicted the growth kinetics and the interaction between the substrates.     

Prevention of <Emphasis Type="Italic">Candida albicans</Emphasis> biofilm by plant oils

The inhibitory effect of 30 plant oils was evaluated against biofilm forming Candida albicans strain (CA I) isolated from clinical samples, which was sensitive to 4 μg/ml of fluconazole, used as a positive control. The standard strain (MTCC 227, CA II) used in this study was found to be highly resistant to fluconazole, 3,000 μg/ml of which was required to inhibit the growth of this strain partially, and complete inhibition could not be achieved. Eighteen among the 30 plant oils tested were found to show anti-Candida activity by disc diffusion assay. Effective plant oils were assessed using XTT (2, 3-bis [2-Methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay for biofilm quantification. Four oils eucalyptus, peppermint, ginger grass and clove showed 80.87%, 74.16%, 40.46% and 28.57% biofilm reduction respectively. Minimum inhibitory concentration (MIC) values were calculated using agar dilution assay. Scanning electron microscopic (SEM) analysis further revealed reduction in C. albicans biofilm in response to effective oils. The substantial antifungal activity shown by these plant oils suggests their potential against infections caused by C. albicans.     

In vitro activity of eugenol against <Emphasis Type="Italic">Candida albicans</Emphasis> biofilms

Most manifestations of candidiasis are associated with biofilm formation occurring on the surfaces of host tissues and medical devices. Candida albicans is the most frequently isolated causative pathogen of candidiasis, and the biofilms display significantly increased levels of resistance to the conventional antifungal agents. Eugenol, the major phenolic component of clove essential oil, possesses potent antifungal activity. The aim of this study was to investigate the effects of eugenol on preformed biofilms, adherent cells, subsequent biofilm formation and cell morphogenesis of C. albicans. Eugenol displayed in vitro activity against C. albicans cells within biofilms, when MIC₅₀ for sessile cells was 500 mg/L. C. albicans adherent cell populations (after 0, 1, 2 and 4 h of adherence) were treated with various concentrations of eugenol (0, 20, 200 and 2,000 mg/L). The extent of subsequent biofilm formation were then assessed with the tetrazolium salt reduction assay. Effect of eugenol on morphogenesis of C. albicans cells was observed by scanning electron microscopy (SEM). The results indicated that the effect of eugenol on adherent cells and subsequent biofilm formation was dependent on the initial adherence time and the concentration of this compound, and that eugenol can inhibit filamentous growth of C. albicans cells. In addition, using human erythrocytes, eugenol showed low hemolytic activity. These results indicated that eugenol displayed potent activity against C. albicans biofilms in vitro with low cytotoxicity and therefore has potential therapeutic implication for biofilm-associated candidal infections.     

Four novel <Emphasis Type="Italic">Candida</Emphasis> species in the <Emphasis Type="Italic">Candida albicans</Emphasis>/<Emphasis Type="Italic">Lodderomyces elongisporus</Emphasis> clade isolated from the gut of flower beetles

Flower-visiting beetles belonging to three species of Cetoniidae were collected on three mountains near Beijing, China, and yeasts were isolated from the gut of the insects collected. Based on the 26S rDNA D1/D2 domain and internal transcribed spacer (ITS) region sequence analysis and phenotypic characterization, four novel anamorphic yeast species located in the Candida albicans/Lodderomyces elongisporus clade were identified from 18 of the strains isolated. The new species and type strains are designated as Candida blackwellae AS 2.3639^T (=CBS 10843^T), Candida jiufengensis AS 2.3688^T (=CBS 10846^T), Candida oxycetoniae AS 2.3656^T (=CBS 10844^T), and Candida pseudojiufengensis AS 2.3693^T (=CBS 10847^T). C. blackwellae sp. nov. was basal to the branch formed by C. albicans and C. dubliniensis with moderately strong bootstrap support. The closest relative of C. oxycetoniae was L. elongisporus. C. jiufengensis sp. nov. and C. pseudojiufengensis sp. nov. were closely related with each other and formed a branch in a subclade represented by C. parapsilosis and L. elongisporus.     

Loss of heterozygosity is induced in <Emphasis Type="Italic">Candida albicans</Emphasis> by ultraviolet irradiation

Candida albicans is a human fungal pathogen and has been extensively studied because of its clinical importance. Comprehensive gene analyses have, however, made little progress. This is because of the diploid and asexual characteristics of the fungus that hamper gene disruptions. In this study, we found that ultraviolet (UV) irradiation, as well as mutagen treatment, strongly stimulated loss of heterozygosity (LOH) in strains harboring artificially constructed heterozygosity. UV-induced LOH occurred more frequently in cells within the logarithmic phase of growth compared to those within the stationary phase of growth. This was observed at all loci tested on chromosome 7, except for a locus neighboring the centromere. C. albicans RAD52, whose orthologue in Saccharomyces cerevisiae was reported to be involved in DNA repair by homologous recombination, was shown to be required for UV-induced LOH. These results suggest that high efficiency LOH caused by UV irradiation could be a prominent tool for gene analyses in C. albicans.     

cAMP regulates vegetative growth and cell cycle in <Emphasis Type="Italic">Candida albicans</Emphasis>

We demonstrate here the regulatory role of cAMP in cell cycle of Candida albicans. cAMP was found to be a positive signal for growth and morphogenesis. Phosphodiesterase inhibitor aminophylline exhibited significant effects, i.e., increased growth, as well as induced morphogenesis. Atropine and trifluoperazine negatively regulated (inhibited) growth and did not induce morphogenesis. These changes were attributed to increase in cAMP levels and protein kinase A (PKA) activity in presence of aminophylline, while reduction was observed in atropine and trifluoperazine (TFP) grown cells. Alteration in cAMP signaling pathway affected the cell cycle progression in Candida albicans. Increased cAMP levels in aminophylline grown cells reduced the duration of cell cycle by inciting the cell cycle-specific expression of G1 cyclins (CLN1 and CLN2). However atropine and trifluoperazine delayed the expression of G1 cyclins and hence prolonged the cell cycle. Implication of cAMP signaling pathway in both the cell cycle and morphogenesis further opened the channels to explore the potential of this pathway to serve as a target for development of new antifungal drugs.     

Inhibition on <Emphasis Type="Italic">Candida albicans</Emphasis> biofilm formation using divalent cation chelators (EDTA)

Candida albicans can readily form biofilms on both inanimate and biological surfaces. In this study we investigated a means of inhibiting biofilm formation using EDTA (Ethylenediaminetetra-acetic acid), a divalent cation chelating agent, which has been shown to affect C. albicans filamentation. Candida albicans biofilms were formed in 96-well microtitre plates. Cells were allowed to adhere for 1, 2, and 4 h at 37°C, washed in PBS, and then treated with different concentrations of EDTA (0, 2.5, 25, and 250 mM). EDTA was also added to the standardized suspension prior to adding to the microtiter plate and to a preformed 24 h biofilm. All plates were then incubated at 37°C for an additional 24 h to allow for biofilm formation. The extent and characteristics of biofilm formation were then microscopically assessed and with a semi-quantitative colorimetric technique based on the use of an XTT-reduction assay. Northern blot analysis of the hyphal wall protein (HWP1) expression was also monitored in planktonic and biofilm cells treated with EDTA. Microscopic analysis and colorimetric readings revealed that filamentation and biofilm formation were inhibited by EDTA in a concentration dependant manner. However, preformed biofilms were minimally affected by EDTA (maximum of 31% reduction at 250 mM). The HWP1 gene expression was reduced in EDTA-treated planktonic and biofilm samples. These results indicate that EDTA inhibits C. albicans biofilm formation are most likely through its inhibitory effect on filamentation and indicates the potential therapeutic effects of EDTA. This compound may serve a non-toxic means of preventing biofilm formation on infections with a C. albicans biofilm etiology.     

Decolorization of textile dye by <Emphasis Type="Italic">Candida albicans</Emphasis> isolated from industrial effluents

The aim of the present work was to observe microbial decolorization and biodegradation of the Direct Violet 51 azo dye by Candida albicans isolated from industrial effluents and study the metabolites formed after degradation. C. albicans was used in the removal of the dye in order to further biosorption and biodegradation at different pH values in aqueous solutions. A comparative study of biodegradation analysis was carried out using UV–vis and FTIR spectroscopy, which revealed significant changes in peak positions when compared to the dye spectrum. Theses changes in dye structure appeared after 72 h at pH 2.50; after 240 h at pH 4.50; and after 280 h at pH 6.50, indicating the different by-products formed during the biodegradation process. Hence, the yeast C. albicans was able to remove the color substance, demonstrating a potential enzymatic capacity to modify the chemical structure of pigments found in industrial effluents.     

Identification and functional characterization of <Emphasis Type="Italic">Candida albicans CDC4</Emphasis>

Summary The CDC4 gene of Saccharomyces cerevisiae encodes an essential function that is required for G1-S and G2-M transitions during mitosis and at various stages during meiosis. We have isolated a functional homologue of CDC4 (CaCDC4) from the pathogenic yeast Candida albicans by complementing the S. cerevisiae cdc4-3 mutation with CaCDC4 expressed from its own promoter on a single-copy vector. The predicted product of CaCDC4 has 37% overall identity to the S. cerevisiae Cdc4 protein, although this identity is biased towards the C-terminal region of the two proteins which contains eight copies of the degenerate WD-40 motif, an element found in proteins that regulate diverse biological processes and an F-box domain proximal to the first iteration of the WD-40 motif. Both the F-box domain and WD-40 motifs appear necessary for the mitotic functions of Cdc4 in both yeasts. In contrast to its conserved role in mitosis, C. albicans CDC4 is unable to rescue the meiotic deficiency in a S. cerevisiae cdc4 homozygous diploid under restrictive conditions, even when expressed from an efficient S. cerevisiae promoter. In opposition to S. cerevisiae CDC4 being essential, C. albicans CDC4 appears to be nonessential and in its absence is critical for filamentous growth in C. albicans.     

Isolation and characterization of microsatellite DNA markers for the flagfish <Emphasis Type="Italic">Jordanella </Emphasis><Emphasis Type="Italic">floridae</Emphasis>

The flagfish (Jordanella floridae) is commonly used in studies of wetlands ecology. Here we describe the isolation of ten microsatellite loci, six of which were polymorphic. The observed number of alleles ranged from 4 to 24 and observed heterozygosities ranged from 0.59 to 0.81 for the polymorphic loci. The isolation of these markers will enable estimations of genetic diversity in natural populations.     

Vulvovaginal candidiasis is associated with the production of germ tubes by <Emphasis Type="Italic">Candida albicans</Emphasis>

Twenty Candida albicans strains isolated from women attended at the Teaching and Research in the Laboratory of Teaching and Research in Clinical Analysis of the State University of Maringa, Paraná, Brazil, have been analyzed. Yeasts were identified by classical methods and patients subdivided into asymptomatic, vulvovaginal candidiasis(VVC) and recurrent vulvovaginal candidiasis (RVVC) groups. Yeasts were incubated in RPMI + fetal calf serum to analyze germ tubes every two hours, up to 10 h. In vitro sensitivity to fluconazole, itraconazole, ketoconazole, amphotericin B and nystatin was analyzed according to NCCLS-M27-A microdilution assay. Yeast isolated from symptomatic women produced significantly more germ tubes than asymptomatic women (P < 0.05). However, no significant difference between yeasts from VVC and RVVC occurred (P > 0.05). Variation between MIC₅₀ and MIC₉₀ of tested antifungal agents was slight among isolated yeasts, while no resistant yeasts were detected. Nevertheless, VVC yeasts were more DDS (reduced dose-dependent susceptibility) for nystatin and RVVC were more DDS for ketoconazole. Results suggest that colonization by yeast in the vagina and lack of symptoms may be partially explained by the yeast’s sparse capacity to form germ tubes, On the other hand, RVVC was not associated with antimicrobial resistance. DDS high frequency for nystatin and ketoconazole indicates that identification, and susceptibility of antifungals tests are important to management of VVC.     

Microsatellite DNA loci from the typical halophyte <Emphasis Type="Italic">Thellungiella salsuginea</Emphasis> (Brassicaceae)

Thellungiella salsuginea (Brassiaceae) is a typical halophyte which can tolerate extreme cold, drought, and salinity. In order to understand the adaptive evolution of this species in the arid habitats, it is important to know its genetic structure. In this study, 17 polymorphic microsatellite loci were isolated and characterized from an enrichment genomic library of this species. We further assessed the polymorphisms of each locus in 18 individuals from nine geographically distant populations. The number of alleles per locus ranged from six to fourteen. The observed and expected heterozygosity ranged from 0.17 to 0.28 and 0.32 to 0.45, respectively. These markers have been crossly checked in another congeneric species, T. halophila. These microsatellite markers will be useful for investigating population genetics and adaptive evolution of this species and morphological divergence between and it and the closely related species.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.