Antonietta Girolamo

Outbreak of Saccharomyces cerevisiae Subtype boulardii Fungemia in Patients Neighboring Those Treated with a Probiotic Preparation of the Organism

ABSTRACT We report an outbreak of Saccharomyces cerevisiae subtype boulardii fungemia among three intensive care unit roommates of patients receiving lyophilized preparations of this fungus. The fungemia was probably due to central venous catheter contamination and resolved after fluconazole treatment. The need for stringent application of proper hygiene when using a probiotic preparation of this organism is emphasized.

In vivo activity of terpinen-4-ol, the main bioactive component of Melaleuca alternifolia Cheel (tea tree) oil against azole-susceptible and -resistant human pathogenic Candida species

BackgroundRecent 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.MethodsOophorectomized, 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.ResultsIn 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.ConclusionOur 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.

Intravaginal and Intranasal Immunizations Are Equally Effective in Inducing Vaginal Antibodies and Conferring Protection against Vaginal Candidiasis

ABSTRACT 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.

Identification of Major Glucan-Associated Cell Wall Proteins of Candida albicans and Their Role in Fluconazole Resistance

ABSTRACT 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.

THE MP65 GENE IS REQUIRED FOR CELL WALL INTEGRITY, ADHERENCE TO EPITHELIAL CELLS AND BIOFILM FORMATION IN CANDIDA ALBICANS

BackgroundThe 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.ResultsThe 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.ConclusionsWe 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.

Glucan-Associated Protein Modulations and Ultrastructural Changes of the Cell Wall in Candida albicans Treated with Micafungin, a Water-Soluble, Lipopeptide Antimycotic

Abstract The composition of glucan-associated proteins (GAP) in the cell wall of Candida albicans was strongly affected by treatment with a sub-MIC yet β-glucan synthesis inhibitory concentration (0.01 μg/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 β1-3 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 β-glucan synthetic machinery but also protein composition and the whole cell wall structure of Candida albicans.

Determination of Legionella pneumophila susceptibility to Melaleuca alternifolia Cheel (tea tree) oil by an improved broth micro-dilution method under vapour controlled conditions

The aim of this study was to determine the in vitro activity of Melaleuca alternifolia Cheel (tea tree) oil (TTO) against 22 strains of Legionella pneumophila of different serogroup and source of isolation. Both a standard broth micro-dilution method, with slight modifications, and a micro-atmosphere diffusion method were used. Furthermore, we have established a simple sealing procedure in the micro-dilution method to determine the antibacterial activity of TTO against Legionella in aqueous phase. The results showed that L. pneumophila, quite irrespective of serogroup and source of isolation, is exquisitely sensitive to TTO, with minimal inhibitory concentration (MIC) ranging from 0.125 to 0.5% v/v, and a bactericidal activity at 0.5% v/v. In addition, we show here that TTO vapours exert critical activity, that must be controlled for reproducible MIC determinations. Overall, our data suggest that TTO could be active as anti-Legionella disinfectant, for control of water system contamination, especially in spas, in small waterlines or in particular respiratory medical devices.

In Vitro Activity of Tea Tree Oil Vaginal Suppositories against Candida spp. and Probiotic Vaginal Microbiota

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 < 2% v/v, they remained viable. TTO‐VS exhibits, in vitro, a selective fungicidal action, slightly affecting only the Bifidobacteriun animalis strain growth belonging to the vaginal microbiota. In vivo studies are needed to confirm the efficacy to prevent acute or recurrent vaginal candidiasis. Copyright