FTIR spectroscopy as a potential tool to analyse structural modifications during morphogenesis of Candida albicans

Candida albicans is a polymorphic organism that grows under certain conditions as blastospores, hyphae or pseudohyphae. The potentials of FTIR spectroscopy for assessing structural differences in C. albicans blastospores and hyphae were investigated. The main observed differences were localised in the polysaccharide (950–1,185 cm⁻¹), protein (1,480–1,720 cm⁻¹), and the fatty acids (2,840–3,000 cm⁻¹) regions. Quantitative evaluation of differences between hyphae and blastospores by curve-fitting of these regions indicate that these modifications could be due to both changes in structure and content of components of the cell wall such as β-glucans, mannoproteins, and lipids. Furthermore, glycogen consumption could be involved during hyphae elongation. Thus, FTIR spectroscopy can be an interesting tool to investigate differences in structure and in content between blastospores and hyphae. We also demonstrate through this study that differentiation of C. albicans clinical strains using hyphae is feasible, as this has been previously shown with blastospores. This preliminary work on identification of C. albicans using hyphae is a prelude to a larger clinical study for early typing within 7 h from a pure culture.     

New milk medium for germ tube and chlamydoconidia production byCandida albicans

A new medium consisting of UHT milk, tween 80 and agar is described for the development of both germ tube and chlamydoconidia byCandida albicans. In total 172 isolates from clinical specimens, includingC. albicans (112),C. guilliermondii (4),C. krusei (3),C. parasilopsis (16).C. tropicalis (28),Torulopsis glabrata (6) andTrichosporon beigellii (3), were examined in this medium by using the standard method. A higher percentage (98.2%) of germ tube production byC. albicans was found in this medium than in undiluted serum (90.2%). In addition, onlyC. albicans was found to be able to produce a high percentage of chlamydoconidia (95.5%) after 48 hours' incubation. In comparison with the conventional medium, corn meal tween 80 agar (21.4%), this new medium gives a significantly higher percentage and abundance of chlamydoconidia production. Being simple, cheap and easy to prepare, the new milk medium is proposed as very practical in the clinical mycology laboratory.     

Utilization of siderophores by Candida albicans

Candida albicans secretes both hydroxamate and phenolate-type siderophores when grown under iron-restricted conditions. The inhibition of candidal growth by iron limitation was reversed by the addition of supplemental hydroxamate on phenolate siderophores. Both siderophores produced equal stimulation of growth suggesting that C. albicans could utilize both siderophores with equal efficiency. Addition of heterologous siderophores from both bacteria and fungi also supported growth of the yeast in a deferrated medium. These results suggest that C. albicans has an iron-uptake mechanism which enables it to obtain iron by utilizing candidal and non-candidal siderophores.     

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.     

A viability assay for Candida albicans based on the electron transfer mediator 2,6-dichlorophenolindophenol

Candida albicans is an opportunistic fungal pathogen with comparably high respiratory activity. Thus, we established a viability test based on 2,6-dichlorophenolindophenol (DCIP), a membrane-permeable electron transfer agent. NADH dehydrogenases catalyze the reduction of DCIP by NADH, and the enzymatic activity can be determined either electrochemically via oxidation reactions of DCIP or photometrically. Among the specific respiratory chain inhibitors, only the complex I inhibitor rotenone decreased the DCIP signal from C. albicans, leaving residual activity of approximately 30%. Thus, the DCIP-reducing activity of C. albicans was largely dependent on complex I activity. C. albicans is closely related to the complex I-negative yeast Saccharomyces cerevisiae, which had previously been used in DCIP viability assays. Via comparative studies, in which we included the pathogenic complex I-negative yeast Candida glabrata, we could define assay conditions that allow a distinction of complex I-negative and -positive organisms. Basal levels of DCIP turnover by S. cerevisiae and C. glabrata were only 30% of those obtained from C. albicans but could be increased to the C. albicans level by adding glucose. No significant increases were observed with galactose. DCIP reduction rates from C. albicans were not further increased by any carbon source.     

The relative contribution of resident pulmonary alveolar macrophage and inflammatory polymorphonuclear neutrophils in host resistance to pulmonary infection by Candida albicans

Cortisone (CA) or cyclophosphamide (Cy) treatment of mice was used to investigate the relative contributions of pulmonary alveolar macrophages (PAM) and inflammatory neutrophils (PMN) in the initial defense against intratracheal challenge (IT) with Candida albicans. Mice treated with either CA or Cy were susceptible to IT challenge with 10–100 x less C. albicans than were untreated mice. Untreated mice rapidly eliminated C. albicans from their lungs with the majority of the organisms being cleared within three hours of challenge. Mice treated with CA initially cleared some of the C. albicans but were unable to clear all the C. albicans as did the untreated mice. Mice treated with Cy were unable to clear C. albicans from their lungs. Candida albicans did not disseminate from the lungs of untreated mice, while in both of the treated groups, C. albicans disseminated to the liver, spleen, brain and kidneys, rapidly killing the treated hosts. Analysis of the changes in cells in lung lavage fluids collected at various times after C. albicans challenge, revealed that large numbers of PMN accumulated in the lungs of both untreated and CA-treated mice, whereas PMN were virtually undetectable in lavage fluids from Cy-treated mice. Resident PAM from untreated mice were able to kill approximately 70 % of 10⁵ C. albicans in a 3 hr in vitro killing assay. By contrast, at similar effector: target ratios, resident PAM from Cy-treated mice killed only about 20% of the inoculum and resident PAM from CA-treated mice were unable to kill C. albicans. PMNs from both untreated and CA-treated mice killed approximately 70% of 10⁵ C. albicans in vitro. The data indicates that both PAM and PMN were critical to the initial clearance of C. albicans from pulmonary tissue. The accumulation of PMN in the lungs appeared to be required for the complete clearance of C. albicans from the lungs yet was not sufficient to inhibit dissemination of C. albicans from the lungs in CA-treated mice. The presence of PAM with in vitro candidacidal abilities appeared to be required for both the clearance of C. albicans and inhibition of dissemination of C. albicans from the lungs. Compromise of either PAM or PMN function can lead to increased pulmonary susceptibility to C. albicans.     

DNA homology between Candida albicans strains: Evidence to justify the synonymous status of C. stellatoidea

Genetic relatedness between strains of C. albicans and C. stellatoidea was studied by measuring G + C content and overall sequence homology. G + C contents determined by high-performance liquid chromatography were 32.6 to 34.2% for 26 strains of C. albicans and 33.0 to 33.9% for eight strains of C. stellatoidea. DNA-DNA hybridization with two C. albicans and two C. stellatoidea probes revealed that all 34 test strains formed a single cluster in which the extents of hybridization with the heterologous probes ranged between 77.9 and 105.6% of those with the homologous probes. These results give support to the unification of C. albicans and C. stellatoidea into a single species.     

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.     

A method for taxonomic determination ofCandida albicans with DNA probes

Determination ofCandida species represents an important problem derived from the clinical implications of the species belonging to this genus. DNA probes have already been used for the epidemiology ofCandida albicans, as well as for taxonomic analysis ofCandida and other genera, although these probes are based on non-species-specific DNA sequences. In this work we carried out a 48-h assay, allowing the identification ofC. albicans from clinical isolates, using DNA probes based onC. albicans LEU2 andURA3 genes. Another probe related toC. albicans SEC18 gene was shown not to beC. albicans specific.     

A simple medium for isolation and identification of Candida albicans directly from clinical specimens

A simple and specific medium consisting of chitosan, trypticase, Tween-80 and agar is devised to isolate the organisms directly from the clinical specimens and to produce germ tubes and chlamydospores for rapid differentiation and identification of Candida albicans from other closely related Candida species. By manipulating the incubating conditions, the specific phase of the organism can be produced in liquid or on solid medium at different time intervals to study the physiology of the organism.Many methods and media have been proposed in the past for identification of Candida albicans and to differentiate this from the closely related species of Candida (5–8, 15). Taschdjian, Burchall&Kozinn (15) showed that C. albicans produces germ tube within an hour or two when it is grown in human or animal serum or serum substitutes. The specificity of this germ tube test was later confirmed by various workers by using different media (3–5). The distinctive feature that differentiates C. albicans from other species is the production of chlamydospores (14). However, in all these studies three types of media were required to isolate the organisms from clinical specimens and to produce germ tubes and chlamydospores for identification. Recently studies have shown that a single medium can be employed to produce both structural components of the organism from the primary isolation medium but the preparation of the medium is more exhaustive (1) and time consuming (13) than the medium to be described here. The present investigation was therefore undertaken to develop a simple and specific medium to isolate the organism directly from the clinical specimens and to produce various morphological phases of Candida albicans to differentiate from other closely related Candida species for clinical diagnosis and to provide a medium to study the physiology and metabolism of the organism under in vitro conditions.Supported in part by Grant CA 20917, National Cancer Institute, National Institutes of Health and ALSAC.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Detection of extracellular phospholipase activity inCandida albicans andRhodotorula rubra

Phospholipases are important pathogenicity determinants inCandida albicans. They play a significant role in damaging cell membranes and invading host cells. High phospholipase production is correlated with an increased ability of adherence and a higher mortality rate in animal models. By means of an egg yolk-containing agar and thePz (= phospholipase activity zone) value according to Price, the present study investigated phospholipase production in 170 strains ofC. albicans. At an incubation temperature of 37 °C,Pz values ranged from 0.395 to 1; no clear relationship was found between clinical origin of the isolates and severity of the disease. In addition toC. albicans, a total of 110 strains of 16 other yeast species were investigated for possible phospholipase production. Only yeasts of the speciesRhodotorula rubra showed phospholipase activity, with mean values exceeding those observed inC. albicans. This result was confirmed by an assay using sterile culture filtrates and phosphatidyl-[3H-methyl]-choline-dipalmitoyl as a substrate. SinceRh. rubra has only rarely been demonstrated as a pathogen in humans, we believe that factors such as reduced growth at 37 °C, absence of dimorphism and low ability of adherence lessen the importance of high phospholipase activity inRh.rubra as a pathogenicity determinant. Therefore, potential virulence factors should always be considered in the context of the whole spectrum of pathogenic determinants.     

A new minimal synthetic medium for germ-tube production in Candida albicans

A new minimal synthetic medium, with low amount of glucose, without aminoacids, vitamins and neutral pH, which induces germ-tubes production in Candida albicans, is reported in this work. The results indicate a perfect agreement between the germ-tube test performed with the standard method in human or animal serum and this test performed in minimal synthetic medium. In this medium the germ-tube test for the presumptive identification of Candida albicans can be performed with the same formality, time and reproducibility as those in human or animal serum. This constitutes an interesting finding because it is easy to prepare, to store and is highly reproducible.     

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.     

Effect of pepstatin A on the virulence factors of Candida albicans strains isolated from vaginal environment of patients in three different clinical conditions

The aspartate proteinase inhibitor pepstatin A was used to study a possible correlation among proteinase activity and other virulence factors of Candida albicans strains isolated from the vaginal environment of patients in three different clinical conditions: asympthomatic, vulvovaginal candidiasis (VVC) and recurrent vulvovaginal candidiasis (RVVC). The addition of 1.0 μM pepstatin A did not have any significant effect on hyphae formation, biofilm production and in the cell surface hydrofobicity of isolates in the three different clinical conditions. However, pepstatin A reduced the adherence of C. albicans to vaginal mucosa epithelial cells (53.1, 48.7 and 59.9%, respectively to isolates from asymptomatic, VVC and RVVC patients). This result suggests that the secreted aspartate proteinases (Saps) of this fungal pathogen may have auxiliary roles in cellular adhesion.     

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.     

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.     

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.     

Effect of Candida albicans cell wall components on the adhesion of the fungus to human and murine vaginal mucosa

In this study, cell walls from Candida albicans were separated and chitin was isolated from these cell walls. A chitin soluble extract (CSE) prepared from the chitin inhibited in vitro adhesion of C. albicans to human epithelial vaginal cells (VEC), and blocked in vivo attachment to murine vaginal mucosa, thereby preventing candidal infection in these animals. These findings suggest that the CSE acts as an adhesin-like substance.Fractionation of CSE yielded two fractions: FI and FII, of which only FI exhibited inhibitory activity. Chemical analysis of CSE and its two fractions revealed that CSE contains over 70% of proteins, most of which were found in the non-active fraction. In addition, 3% of amino-sugars were found in the FI active fraction. Lipids were also detected in the unfractionated CSE and in both fractions.Experiments to further characterize the component(s) in the CSE inhibiting the attachment of C. albicans are in progress in our laboratory.     

Detection of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">dubliniensis</Emphasis> in Venezuela

Over the past decades there has been a significant increase in fungal infections caused by Candida species, and continues to be common in immunocompromised individuals infected with the human immunodeficiency virus (HIV). Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other non-albicans are often identified in this cohort of patients, including C. dubliniensis. This yeast is closely related to and shares many phenotypic characteristics with C. albicans. Colonies of these two species appear morphologically identical when not grown on special media. The shared phenotypic characteristics of C. dubliniensis and C. albicans suggest that many C. dubliniensis isolates may have been misidentified as C. albicans in the past. The present studies aim is to recover and identify C. dubliniensis, and presumptive clinical C. albicans, from the oral cavities of HIV-seropositive individuals, comparing conventional media to obtain a simple, low-cost and reliable identification system for C. dubliniensis. A total of 16 isolates (3,98%) had been obtained from 402 HIV infected individuals with recurrent oropharyngitis and were identified as C. dubliniensis. Out of these C. dubliniensis isolates 19% were resistant, with MICs above 64 μg/ml to fluconazole. This constitutes, to the authors knowledge the first recovery of this organism in Venezuela.