Critical role of germ tube formation in the pathogenesis of candidal vaginitis.

A variant strain of Candida albicans incapable of hyphal production at 37 degrees C was used to study the role of germ tube formation in the pathogenesis of experimental vaginal candidiasis in rats. No difference was observed in the in vitro adherence at 25 degrees C of blastoconidia of the variant strain to vaginal epithelial cells when compared with the parent wild-type, germ tube-producing strain and multiple clinical isolates of C. albicans. However, after exposure to conditions favoring germ tube production, the adherence of the variant strain to epithelial cells was significantly less than that of germinated strains (P less than 0.01). In vivo animal studies revealed that the variant strain was less likely to result in vaginal colonization and infection than the wild-type strain and the other clinical isolates. Furthermore, infection, when established, was milder, often transient, and with significantly lower titers of cultured vaginal microorganisms obtained by lavage. Electron microscopic studies confirmed the failure of the variant strain to produce hyphae in vivo. The capacity of C. albicans to produce hyphae appears to be an important but nonessential virulence factor in the pathogenesis of candidal vaginitis.     

Modulation of cell surface-associated mannoprotein antigen expression in experimental candidal vaginitis.

The monoclonal antibody (MAb) AF1 recognizes an oligosaccharide epitope present on highly immunogenic and immunomodulatory mannoproteins (MP) of Candida albicans. The expression of this epitope (AF1-MP) during experimental candidal vaginitis was studied in two strains of C. albicans (3153 and CA-2) which were equally vaginopathic but differed in the mode of hypha formation in the vagina. In both strains, immunofluorescence of vaginal samples, taken 1 h after challenge, revealed an intense, MAb AF1-specific labelling of the yeast cells. This labelling was very scarce in fungal cells taken at 24 h and on subsequent days during the development of filamentous forms. Electron-microscopic gold immunolabelling observations showed that molecules carrying AF1-MP spanned the entire cell wall in the initial yeast cells but were absent on the cell surface and in the outermost, capsular layer of the cell wall of the germ tubes and filamentous forms. In both strains, at any time and for any form of intravaginal growth, AF1-MP was clearly expressed in the cytoplasm and cytoplasmic vesicles, and was fully incorporated into the inner layers of the cell wall. As seen by immunofluorescence, the vaginal fluid from C. albicans-infected rats did not hinder the expression of AF1-MP on the yeast cells surface in vitro. In electron-microscopic gold immunolabelling, a hypha-specific MAb (3D9) labelled the surface of the hyphal but not of the yeast cells of C. albicans harvested from rat vagina. Overall, these data strongly suggest that cell surface expression of MP antigen is modulated during intravaginal growth and morphogenesis of C. albicans.     

Genotoxicity of lavender oil,linalyl acetate,and linalool on human lymphocytes in vitro

The potential genotoxicity of lavender essential oil and its major components, linalool, and linalyl acetate, was evaluated in vitro by the micronucleus test on peripheral human lymphocytes. In the range of non‐toxic concentrations (0.5–100 μg/ml), linalyl acetate increased the frequency of micronuclei significantly and in concentration‐dependent manner; lavender oil did so only at the highest concentration tested, whereas linalool was devoid of genotoxicity. None of the tested substances led to an increase in nucleoplasmic bridges or nuclear buds frequency. These findings suggest that the mutagenic activity of lavender oil can be related to the presence of linalyl acetate, which seems to have a profile of an aneugenic agent. Environ. Mol. Mutagen. 52:69–71, 2011. © 2010 Wiley‐Liss, Inc.     

In vitro effect of fibrinogen on Candida albicans germ tube formation

Pseudohyphae formation by Candida albicans blastoconidia, as seen in vaginal smears, is a phenotypical change commonly assumed to mean fungal invasiveness, i.e. not mere colonization. C. albicans forms germ tubes in vitro in the presence of serum. In our search for inhibitory components of germ tube formation, we decided to study fibrinogen. The inhibition of germ tube formation by clinical isolates of C. albicans was evaluated in the presence of serial concentrations of fraction I, type IV and fraction I, type Is of fibrinogen from bovine plasma. Fibrinogen showed a dose-dependent, pH-independent inhibitory effect on the germ tube formation by C. albicans.     

Detection of human P-glycoprotein-like molecule in azole-resistant Candida albicans from HIV+ patients

Azole resistance in Candida albicans may be due to several mechanisms. It has been demonstrated that C. albicans possesses sequences with a high degree of homology with the human MDR-1 gene coding for P-glycoprotein (P-gp), belonging to the ATP-binding cassette transporter (ABC) superfamily and responsible for the multidrug resistance (MDR) in tumor cells. On this basis, the expression and intracellular localization of human P-gp-like molecule in C. albicans strains showing different sensitivity to fluconazole were investigated by flow cytometry and immunoelectron microscopy. Post-embedding immunolabeling revealed that monoclonal antibody (mAb) MM4.17, which recognizes an external epitope of human P-gp, reacted with both fluconazole-sensitive (3153 and CO 23-1) and fluconazole-resistant (AIDS 68 and CO 23-2, isolated from AIDS patient and in vitro drug-selected, respectively) strains of C. albicans. However, the resistant strains displayed a number of MM4.17-reactive epitopes much higher than the drug-sensitive ones. The C. krusei ATCC 6458 strain, whose resistance is not mediated by the presence of ABC transporters, was not reactive at all with mAb MM4.17. The specificity of the immunolabeling was confirmed by a competitive inhibition assay performed by using phage clone particles capable of mimicking the MM4.17-reactive epitope. The flow cytometric analysis confirmed a higher level of intracytoplasmic P-gp expression in azole-resistant strains of C. albicans. Both cyclosporin A and verapamil, which are well-known MDR inhibitors, strongly reduced the MICs for fluconazole and itraconazole of the tested azole-resistant AIDS 68 strain, while they did not influence the MICs of either the sensitive 3153 strain of C. albicans or the ATCC 6458 strain of C. krusei. Overall, our data suggest the existence of a P-gp-like drug efflux pump in C. albicans that may participate in the mechanisms of azole-resistance of this fungus.     

Structure and regulation of the HSP90 gene from the pathogenic fungus Candida albicans.

Candida albicans HSP90 sequences were isolated by screening cDNA and genomic libraries with a probe derived from the Saccharomyces cerevisiae homolog, HSP82, which encodes a member of the heat shock protein 90 family of molecular chaperones. Identical sequences were obtained for the 2,197-bp overlap of the cDNA and gene sequences, which were derived from C. albicans 3153A and ATCC 10261, respectively. The C. albicans HSP90 gene contained no introns, and it showed strong homology (61 to 79% identity) to HSP90 sequences from other fungi, vertebrates, and plants. The C-terminal portion of the predicted Hsp90 amino acid sequence was identical to the 47-kDa protein which is thought to be immunoprotective during C. albicans infections (R. C. Matthews, J. Med. Microbiol. 36:367-370, 1992), confirming that this protein represents the C-terminal portion of the 81-kDa Hsp90 protein. Quantitative Northern (RNA) analyses revealed that C. albicans HSP90 mRNA was heat shock inducible and that its levels changed during batch growth, with its maximum levels being reached during the mid-exponential growth phase. HSP90 mRNA levels increased transiently during the yeast-to-hyphal transition but did not correlate directly with germ tube production per se. These data do not exclude a role for Hsp90 in the dimorphic transition. Southern blotting revealed only one HSP90 locus in the diploid C. albicans genome. Repeated attempts to disrupt both alleles and generate a homozygous C. albicans delta hsp90/delta hsp90 null mutant were unsuccessful. These observations suggest the existence of a single HSP90 locus which is essential for viability in C. albicans.     

Melaleuca alternifolia (tea tree) oil inhibits germ tube formation by Candida albicans.

The effect of tea tree oil (TTO) on the formation of germ tubes by Candida albicans was examined. Two isolates were tested for germ tube formation (GTF) in the presence of TTO concentrations (% v/v) ranging from 0.25% (1/2 minimum inhibitory concentration [MIC]) to 0.004% (1/128 MIC). GTF at 4 h in the presence of 0.004 and 0.008% (both isolates) and 0.016% (one isolate) TTO did not differ significantly (P > 0.05) from controls. At all other concentrations at 4 h, GTF differed significantly from controls (P < 0.01). A further eight isolates were tested for GTF in the presence of 0.031% TTO, and at 4h the mean GTF for all 10 isolates ranged 10.0-68.5%. Two isolates were examined for their ability to form germ tubes after 1 h of pre-exposure to several concentrations of TTO, prior to induction of germ tubes in horse serum. Cells pre-exposed to 0.125 and 0.25% TTO formed significantly fewer germ tubes than control cells at 1 h (P < 0.05), but only those cells pre-exposed to 0.25% differed significantly from control cells at later time points (P < 0.01). GTF by C. albicans is affected by the presence of, or pre-exposure to, sub-inhibitory concentrations of TTO. This may have therapeutic implications.     

In vitro investigation of antifungal activities of phenotypic variation Candida albicans strains against fluconazole, itraconazole and voriconazole.

The aim of this study was to investigate the susceptibility of the different phenotypes of Candida albicans strains isolated from clinical specimens to three antifungal agents, fluconazole, itraconazole and voriconazole. Totally 215 specimens were collected from oropharyngeal, gastrointestinal and urogenital tracts of non-neutropenic patients who had received no previous prophylactic treatment. Each of the 215 C. albicans strains recovered was found to express one of the six phenotypes: smooth 73%, fuzzy 10.7%, irregular 2.3%, star 2.8%, ring 6% or stipple 5.1%. The mean MICs for the six phenotypes of C. albicans strains ranged between 0.25 microg/ml and 64 microg/ml for fluconazole, 0.03 microg/ml and 1 microg/ml for itraconazole and 0.03 microg/ml and 0.5 microg/ml for voriconazole. The mean minimum inhibitory concentration (MIC) of fluconazole was consistently higher for C. albicans strains expressing the stipple phenotype. The antifungal susceptibility of the phenotypic switching requires attention, especially in patients who are clinically unresponsive to fluconazole chemotherapy or in cases of serious C. albicans infections of immunocompromised hosts.     

Hormonal factors in vaginal candidiasis in rats.

The hormonal status of rats affected vaginal infection with Candida albicans. Four hours after infection viable counts were higher and germ tubes were longer in those animals in estrous than in other animals. However, the infection was not maintained with the change in epithelial cell type which occurred as part of the estrous cycle. Estrogen dosing following ovariectomy predisposed toward infection, while progesterone dosing did not. In rats injected with progesterone, germ tube clumping was seen, leukocytes were present, and elimination occurred before hyphal growth was evident. In rats injected with estrogen, however, infection was maintained, with hyphal growth extending throughout the cornified epithelial layer. Vaginal washings from rats dosed with estrogen promoted elongation of germ tubes in vitro to a greater extent than washings from other rats. Preincubation of blastospores in progesterone and subsequent infection of rats in pseudoestrous promoted clumping of germ tubes in the vagina. Strains of C. albicans varied in their virulence, which correlated with their ability to produce germ tubes in vitro. Loss of virulence occurred on subculture of a clinical isolate.     

Use of a commercial reagent leads to reduced germ tube production by Candida dubliniensis

The goal of this study was to determine the factor(s) explaining our inability to detect Candida dubliniensis. When germ tube-positive yeasts were tested for C. dubliniensis, no C. dubliniensis was detected; however, 58 C. dubliniensis strains were detected when germ tube-negative Candida albicans strains were tested further. Since all 58 C. dubliniensis strains detected were germ tube negative, these data implied that false-negative germ tube tests occurred with germ tube solution (GTS; Remel, Lenexa, KS). All 41 known C. dubliniensis strains tested were negative with GTS, whereas 40 were positive with rabbit serum (RS; Sigma-Aldrich, St. Louis, MO). Results for C. albicans were equivalent in GTS and RS. In conclusion, GTS cannot be used for the detection of C. dubliniensis, and switching from yeast to hyphae in C. dubliniensis is more restricted than in C. albicans.     

Isoenzyme changes in Candida albicans during domestication.

Isoenzyme analysis was performed on multiple strains of two commonly used reference cultures of Candida albicans (B311 and NCPF3153). Whereas strains originating from C. albicans B311 showed no variation in isoenzyme profiles, some strains derived from NCPF3153 were identical to B311 strains but others showed variation in their glucose-6-phosphate dehydrogenase isoenzymes. The results are compared with those from previous analyses with these strains and show that C. albicans can undergo genetic alterations during prolonged maintenance in laboratories.     

Fab fragments from a monoclonal antibody against a germ tube mannoprotein block the yeast-to-mycelium transition in Candida albicans.

Fab fragments prepared from the immunoglobulin G monoclonal antibody (MAb) 4C12, which reacts with a determinant expressed on the hyphal extension of germ tubes of Candida albicans, inhibited germ tube formation, but intact MAb 4C12 did not. Indirect immunofluorescence showed a punctate binding pattern on cells incubated with Fab fragments but a confluent binding on cells incubated with intact MAb 4C12.     

Selection of candidate quality control isolates and tentative quality control ranges for in vitro susceptibility testing of yeast isolates by National Committee for Clinical Laboratory Standards proposed standard methods.

The National Committee for Clinical Laboratory Standards has developed a proposed standard method for in vitro antifungal susceptibility testing of yeast isolates (National Committee for Clinical Laboratory Standards, document M27-P, 1992). In order for antifungal testing by the M27-P method to be accepted, reliable quality control (QC) performance criteria must be developed. In the present study, five laboratories tested 10 candidate QC strains 20 times each against three antifungal agents: amphotericin B, fluconazole, and 5-fluorocytosine. All sites conformed to the M27-P standards and used a common lot of tube dilution reagents and RPMI 1640 broth medium. Overall, 98% of MIC results with amphotericin B, 95% with fluconazole, and 92% with 5-fluorocytosine fell within the desired 3-log2 dilution range (mode +/- 1 log2 dilution). Excellent performance with all three antifungal agents was observed for six strains: Candida albicans ATCC 90028, Candida parapsilosis ATCC 90018, C. parapsilosis ATCC 22019, Candida krusei ATCC 6258, Candida tropicalis ATCC 750, and Saccharomyces cerevisiae ATCC 9763. With these strains, 3-log2 dilution ranges encompassing 94 to 100% of MICs for all three drugs were established. Additional studies with multiple lots of RPMI 1640 test medium will be required to establish definitive QC ranges.     

Regulation of Candida albicans morphogenesis by fatty acid metabolites

Candida albicans is an opportunistic dimorphic fungus that inhabits various host mucosal sites. Conversion from the yeast to the hyphal form has been associated with increased virulence and mucosal invasiveness. C. albicans morphogenesis is regulated by multiple signals and signaling pathways. However, signals that control morphogenesis in vivo are unknown. We investigated the effects of host long chain fatty acids, eicosanoids, and bacterial short chain fatty acids on control of germination. None of the C18 or C20 fatty acids tested had an effect on enhancing germ tube formation (arachidonic acid, oleic acid, linolenic acid, or gamma-linolenic acid). Among the different eicosanoids, both prostaglandin E2 and thromboxane B2 significantly enhanced serum-induced germination by C. albicans. Addition of antiprostaglandin or antithromboxane antibodies to serum alone inhibited germ tube formation by almost 30%, while control antibody had no effect, indicating that these eicosanoids are major morphogenic factors in the serum. Since these molecules also bind to albumin, this may also explain the hyphal transforming activity in serum that associates with albumin. Interestingly, short chain fatty acids (butyric acid), the product of lactic acid bacteria (LAB), inhibited germination. In addition, LAB culture supernatants as well as live LAB also inhibited C. albicans morphogenesis. Overall, these results indicate that fatty acid metabolites and fatty acid pathways can up-regulate and down-regulate germination in C. albicans.     

Antifungal activity of Ferulago capillaris essential oil against Candida, Cryptococcus, Aspergillus and dermatophyte species

This study evaluates the composition, antifungal activity and mechanism of action of the essential oil of Ferulago capillaris (Link ex Spreng.) Cout. and its main components, limonene and α-pinene, against clinically relevant yeasts and moulds. Essential oil from the plant’s aerial parts was obtained by hydrodistillation and analysed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC-MS). Essential oil showed high contents of limonene (30.9 %) and α-pinene (35.8 %). Minimum inhibitory concentrations (MICs) were measured according to the reference Clinical and Laboratory Standards Institute (CLSI) broth macrodilution protocols. Cell suspensions were subcultured in solid medium and the minimum fungicidal concentrations (MFCs) were rendered. The effect of essential oil on germ tube formation, mitochondrial function and ergosterol biosynthesis was investigated. Essential oil and α-pinene displayed low and similar MIC and MFC values against tested organisms (0.08 to 5.0 μL/mL), while limonene showed a weaker activity (0.32 to 20 μL/mL). Essential oil inhibited germ tube formation at sub-inhibitory concentrations on Candida albicans. The exposure of C. albicans to the essential oil resulted in impairment of mitochondrial functions in a dose-dependent manner. No difference in ergosterol content was observed in essential oil-treated C. albicans. F. capillaris and α-pinene display a broad fungicidal activity. The fungicidal activity of F. capillaris on C. albicans can be related to an induced oxidative stress which affects enzymes activity and the membrane potential of mitochondria. The essential oil of F. capillaris was shown to have potential for use in the development of clinically useful therapeutic preparations, particularly for topical application in the management of superficial mycoses.     

Variations in fluconazole susceptibility and electrophoretic karyotype among oral isolates of Candida albicans from patients with AIDS and oral candidiasis.

DNA subtyping by pulsed-field gel electrophoresis and in vitro susceptibility testing were used to study strain variation and fluconazole resistance in Candida albicans isolates from patients with AIDS undergoing azole (fluconazole and clotrimazole) therapy for oropharyngeal candidiasis. A total of 29 patients suffered 71 episodes of oropharyngeal candidiasis. Overall, 121 isolates of C. albicans recovered throughout the course of treatment of each infection were available for further characterization. DNA subtyping revealed a total of 61 different DNA subtypes. In vitro susceptibility testing of the 121 isolates by using proposed standard methods of the National Committee for Clinical Laboratory Standards revealed MICs of fluconazole ranging from < or = 0.125 to > 64 micrograms/ml. The MIC for 50% of isolates tested was 0.25 microgram/ml, and the MIC for 90% of isolates tested was 8.0 micrograms/ml. MICs were > or = 64 micrograms/ml for only 7.4% of the isolates tested. The majority (62%) of the patients with oropharyngeal candidiasis and undergoing azole therapy were infected or colonized with more than one DNA subtype, and the introduction or selection of strains with a more resistant DNA subtype during the course of fluconazole therapy was not uncommon. With one exception, this did not appear to have an adverse effect on clinical outcome. In contrast, for patients with AIDS and oropharyngeal candidiasis infected with a single DNA subtype of C. albicans, an increase in fluconazole MICs for the infecting strain was rarely demonstrated over the course of therapy.     

Defective hyphal development and avirulence caused by a deletion of the SSK1 response regulator gene in Candida albicans

In a previous study, we reported the isolation and characterization of the two-component response regulator SSK1 gene of Candida albicans. This gene is a structural but not a functional homolog of the SSK1 and mcs4(+) genes of Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. In the present study, we have constructed and phenotypically characterized Deltassk1 mutants of C. albicans. The results confirmed our previous observation that CaSSK1, unlike SSK1 or mcs4(+), does not regulate cellular responses to either osmotic or oxidative stress. Instead, Deltassk1 null strains showed severely reduced hyphal formation on serum agar and were totally defective in hyphal development on other solid media, such as medium 199 (pH 7.5) and Spider medium. In contrast, under conditions of low nitrogen availability on solid media, Deltassk1 null strains dramatically hyperinvaded the agar. However, while forming germ tubes and hyphae in liquid media similar to those of the wild type, Deltassk1 null strains flocculated in a manner similar to that of Deltachk1 two-component histidine kinase mutants, which we have previously described. Finally, virulence studies indicated that SSK1 is essential for the pathogenesis of C. albicans, suggesting that the Ssk1p response regulator could be a good target for antifungal therapy.     

Prevalence of Candida dubliniensis in the BCCM/IHEM Biomedical Fungi/Yeasts culture collection (isolates before 1990).

The BCCM/IHEM Biomedical Fungi/Yeasts collection hosts 1200 Candida albicans strains of the Vanbreuseghem mycotheque isolated between 1951 and 1997. From this collection, 469 freeze-dried C. albicans strains, producing chlamydospores, germ tubes and forming green colonies on CHROMagar, all isolated before 1990, were screened to identify the Candida dubliniensis isolates. Screening was performed in different steps using the growth at 45 degrees C, the assimilation of xylose, the intracellular beta-glucosidase activity test and C. dubliniensis-specific polymerase chain reaction (PCR) with primers from ACT1 intron sequence. Five isolates (1%) were identified as C. dubliniensis: one isolate was not documented, the others were of oropharyngeal origin of which two (1987 and 1990) were from proven human immunodeficiency virus patients.     

Discrimination between Candida albicans and other pathogenic species of the genus Candida by their differential sensitivities to toxins of a panel of killer yeasts

The differential sensitivities to toxins produced by a short panel of four killer yeasts allowed discrimination between 91 strains of the yeast Candida albicans and 223 non-C. albicans Candida strains. One hundred percent of C. albicans isolates exhibited negative results to the toxin panel, while 100% of non-C. albicans cultures gave well-defined and reproducible positive results to at least one of the four killer toxins. Among C. albicans strains only 96 and 87% gave germ tube (GT)- and chlamydospore-positive results, respectively. In addition a few GT-false-positive strains were detected among non-C. albicans isolates. Susceptibility to the toxin panel is apparently expressed more consistently than either GT or chlamydospore production and may constitute a promising basis for a new simple and easy-to-use procedure for routine discrimination between the species C. albicans and other species of the genus Candida.