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The aim of this work is to evaluate the in vitro microbicidal activity of vaginal suppositories (VS) containing tea tree oil (TTO-VS) towards Candida spp. and vaginal probiotics. A total of 20 Candida spp. strains, taken from patients with vaginitis and from an established type collection, including reference strains, were analysed by using the CLSI microdilution method. To study the action of VS towards the beneficial vaginal microbiota, the sensitivity of Bifidobacterium animalis subsp. lactis (DSM 10140) and Lactobacillus spp. (Lactobacillus casei R-215 and Lactobacillus acidophilus R-52) was tested. Both TTO-VS and TTO showed fungicidal activity against all strains of Candida spp. whereas placebo-VS or the Aloe gel used as controls were ineffective. The study of fractional fungicidal concentrations (FFC) showed synergistic interaction with the association between Amphotericin B and TTO (0.25 to 0.08 µg/ml, respectively) against Candida albicans. Instead, the probiotics were only affected by TTO concentration ≥ 4% v/v, while, at concentrations

The composition of glucan-associated proteins (GAP) in the cell wall of Candida albicans was strongly affected by treatment with a sub-MIC yet beta-glucan synthesis inhibitory concentration (0.01 microg/ml) of FK463 (micafungin). Namely, a decrease in enzymes of glucose metabolism (mostly enolase and a novel 40 kDaltons component, here identified as the enzyme fructose-1,6-biphosphate aldolase) was observed, and this was coupled with an increase in two beta1-3 exo-glucanase isoforms (34 and 44 kDa, respectively). No GAP changes were detected in the same strain of the fungus made resistant to the drug, attesting to the specificity of the observed cell wall protein modulation. In addition, GAP changes were accompanied by marked ultrastructural alterations upon treatment with the sub-MIC dose of the drug, the majority of which was an aberrant cell surface morphology and a derangement of the normal layering of the cell wall. Our data demonstrate that sub-MIC doses of micafungin do critically affect not only the beta-glucan synthetic machinery but also protein composition and the whole cell wall structure of Candida albicans.

Identification of major glucan-associated proteins (GAPs) of the cell wall of a number of Candida albicans isolates susceptible or resistant to fluconazole (FLC) was addressed by direct sequencing of the protein bands resolved by unidimensional gel electrophoresis. Changes in the GAP compositions of the different strains grown in the presence of the drug were also investigated. In the FLC-susceptible strains, the major (more abundant) GAPs were enolase (46 kDa), two isoforms of phosphoglyceromutase (32 and 29 kDa), and two β-(1-3)-exoglucanases (44 and 34 kDa), one of which (the 34-kDa component) was glycosylated. When these strains were grown in the presence of FLC there were substantial decreases in the intensities of the two enzymes of the glycolytic pathway (enolase and the phosphoglyceromutases), which were apparently replaced by enhancement of the exoglucanase constituents, particularly the 44-kDa one. This GAP pattern closely mimicked that observed in the FLC-resistant strains whether they were grown in the presence or in the absence of the drug. Both the enolase and the exoglucanase constituents were detected in the culture supernatants of FLC-treated cells, together with substantial amounts of highly glycosylated, probably mannoprotein secretory material, suggesting that FLC may cause marked alterations of GAP incorporation into the cell wall. Altogether, we were able to identify all major GAP constituents and monitor their distributions in the cell wall of C. albicans during treatment with FLC. The near equivalence of the GAP profile for the FLC-susceptible strain grown in the presence of FLC to that for the FLC-resistant strain suggests that the effects of the drug on GAPs may be stably incorporated into the cell wall of the fungus upon acquisition of resistance.

Oophorectomized, estrogen-treated rats were immunized by the intravaginal or intranasal route with a mannoprotein extract (MP) or secreted aspartyl proteinases (Sap) of Candida albicans , with or without cholera toxin as a mucosal adjuvant. Both routes of immunization were equally effective in (i) inducing anti-MP and anti-Sap vaginal antibodies and (ii) conferring a high degree of protection against the vaginal infection by the fungus. These data suggest that appropriate fungal antigens and adjuvant can be used to protect against candidal vaginitis, by either route.

Background The MP65 gene of Candida albicans (orf19.1779) encodes a putative β-glucanase mannoprotein of 65 kDa, which plays a main role in a host-fungus relationship, morphogenesis and pathogenicity. In this study, we performed an extensive analysis of a mp65Δ mutant to assess the role of this protein in cell wall integrity, adherence to epithelial cells and biofilm formation. Results The mp65Δ mutant showed a high sensitivity to a range of cell wall-perturbing and degrading agents, especially Congo red, which induced morphological changes such as swelling, clumping and formation of hyphae. The mp65Δ mutant showed an activation of two MAPKs (Mkc1p and Cek1p), a high level of expression of two stress-related genes (DDR48 and SOD5), and a modulated expression of β-glucan epitopes, but no gross changes in cell wall polysaccharide composition. Interestingly, the mp65Δ mutant displayed a marked reduction in adhesion to BEC and Caco-2 cells and severe defects in biofilm formation when compared to the wild type. All of the mentioned properties were totally or partially recovered in a revertant strain, demonstrating the specificity of gene deletion. Conclusions We demonstrate that the MP65 gene of Candida albicans plays a significant role in maintaining cell wall integrity, as well as in adherence to epithelia and biofilm formation, which are major virulence attributes of this fungus.

Background Recent investigations on the antifungal properties of essential oil of Melaleuca alternifolia Cheel (Tea Tree Oil, TTO) have been performed with reference to the treatment of vaginal candidiasis. However, there is a lack of in vivo data supporting in vitro results, especially regarding the antifungal properties of TTO constituents. Thus, the aim of our study was to investigate the in vitro and the in vivo anti-Candida activity of two critical bioactive constituents of TTO, terpinen-4-ol and 1,8-cineole. Methods Oophorectomized, pseudoestrus rats under estrogen treatment were used for experimental vaginal infection with azole (fluconazole, itraconazole) -susceptible or -resistant strains of C. albicans. All these strains were preliminarily tested for in vitro susceptibility to TTO, terpinen-4-ol and 1,8-cineole for their antifungal properties, using a modification of the CLSI (formerly NCCLS) reference M27-A2 broth micro-dilution method. Results In vitro minimal inhibitory concentrations (MIC90) values were 0.06% (volume/volume) for terpinen-4-ol and 4% (volume/volume) for 1,8-cineole, regardless of susceptibility or resistance of the strains to fluconazole and itraconazole. Fungicidal concentrations of terpinen-4-ol were equivalent to the candidastatic activity. In the rat vaginal infection model, terpinen-4-ol was as active as TTO in accelerating clearance from the vagina of all Candida strains examined. Conclusion Our data suggest that terpinen-4-ol is a likely mediator of the in vitro and in vivo activity of TTO. This is the first in vivo demonstration that terpinen-4-ol could control C. albicans vaginal infections. The purified compound holds promise for the treatment of vaginal candidiasis, and particularly the azole-resistant forms.

A tea tree oil (TTO) preparation of defined chemical composition was studied, using a micro- broth method, for its in vitro activity against 115 isolates of Candida albicans, other Candida species and Cryptococcus neoformans. The fungal strains were from HIV-seropositive subjects, or from an established type collection, including reference and quality control strains. Fourteen strains of C. albicans resistant to fluconazole and/or itraconazole were also assessed. The same preparation was also tested in an experimental vaginal infection using fluconazole- itraconazole-susceptible or -resistant strains of C. albicans. TTO was shown to be active in vitro against all tested strains, with MICs ranging from 0.03% (for C. neoformans) to 0.25% (for some strains of C. albicans and other Candida species). Fluconazole- and/or itraconazole-resistant C. albicans isolates had TTO MIC 50 s and MIC 90 s of 0.25% and 0.5%, respectively. TTO was highly efficacious in accelerating C. albicans clearance from experimentally infected rat vagina. Three post-challenge doses of TTO (5%) brought about resolution of infection regardless of whether the infecting C. albicans strain was susceptible or resistant to fluconazole. Overall, the use of a reliable animal model of infection has confirmed and extended our data on the therapeutic effectiveness of TTO against fungi, in particular against C. albicans.

Vaginal isolates of Candida albicans from human immunodeficiency virus-positive (HIV + ) and HIV − women with or without candidal vaginitis were examined for secretory aspartyl proteinase (Sap) production in vitro and in vivo and for the possible correlation of Sap production with pathology and antimycotic susceptibility in vitro. HIV + women with candidal vaginitis were infected by strains of C. albicans showing significantly higher levels of Sap, a virulence enzyme, than strains isolated from HIV + , C. albicans carrier subjects and HIV − subjects with vaginitis. The greater production of Sap in vitro was paralleled by greater amounts of Sap in the vaginal fluids of infected subjects. In an estrogen-dependent, rat vaginitis model, a strain of C. albicans producing a high level of Sap that was isolated from an HIV + woman with vaginitis was more pathogenic than a strain of C. albicans that was isolated primarily from an HIV − , Candida carrier. In the same model, pepstatin A, a strong Sap inhibitor, exerted a strong curative effect on experimental vaginitis. No correlation was found between Sap production and antimycotic susceptibility, as most of the isolates were fully susceptible to fluconazole, itraconazole, and other antimycotics, regardless of their source (subjects infected with strains producing high or low levels of Sap, subjects with vaginitis or carrier subjects, or subjects with or without HIV). Thus, high Sap production is associated with virulence of C. albicans but not with fungal resistance to fluconazole in HIV-infected subjects, and Sap is a potentially new therapeutic target in candidal vaginitis.