Francesca Mondello

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.

Vaginopathic and proteolytic Candida species in outpatients attending a gynaecology clinic.

Non-pregnant, non-diabetic outpatients were examined for the presence of pathogenic vaginal yeasts to determine if a correlation existed between a specific yeast and clinical disease. Yeasts were isolated as single vaginal species from 186 of 228 subjects with clinically diagnosed candidal vaginitis, as well as from 122 out of 380 asymptomatic, age-matched controls. Apart from Candida albicans and C glabrata, other prevalent species were C krusei, C parapsilosis and Saccharomyces cerevisiae which accounted for 9.2%, 6.0% and 5.4%, and 9.0%, 2.4% and 19.7%, of yeasts from patients and carriers, respectively. Only C albicans and C parapsilosis were significantly more common in those with vaginitis. Only the isolates of these two species secreted aspartyl proteinase in vitro, and the amount of the enzymes secreted by the isolates from patients was significantly higher than that secreted by the isolates from carriers. These two species consistently produced vaginal infection in pseudoestrus rats, whereas none of the non-proteolytic species tested (C glabrata, C krusei, and S cerevisiae) colonised the vagina in these rats. Proteinase secretion correlated with experimental vaginal infection; it could also be a reliable factor for distinguishing clinically active infection from asymptomatic fungal carriage.

Generation of a Recombinant 65-Kilodalton Mannoprotein, a Major Antigen Target of Cell-Mediated Immune Response to Candida albicans

ABSTRACT A 65-kDa mannoprotein (CaMp65) has long been studied as a major, immunodominant antigen of the human opportunistic pathogenCandida albicans. An expression library of C. albicans was screened with serum from mice immunized with ScMp65 (ScW10), a Saccharomyces cerevisiae recombinant protein of about 48 kDa. This serum recognized the CaMp65 from a cell wall extract of C. albicans. After cloning and sequencing of the relevant C. albicans cDNA, an open reading frame encoding a protein of 379 amino acids was identified. Its deduced amino acid sequence showed regions of identity with all previously characterized tryptic fragments of CaMp65, as well as with the corresponding regions of ScMp65. A prepeptide of 32 amino acids with signal peptidase and Kex2 cleavage sites as well as a high number of potential O-glycosylation sites but no N-glycosylation sites or GPI anchor were observed in sequence studies of CaMp65. A putative adhesin RGD sequence was also present in the C-terminal region of the molecule. This triplet was absent in the ScMp65. The relevant gene (designatedCaMP65) was localized to chromosome R of C. albicans as determined by pulse-field gel electrophoresis. Northern blot analysis demonstrated that gene transcription was heat inducible and associated with germ-tube formation by the fungus. A recombinant, His6-tagged protein (rCaMp65) was expressed inEscherichia coli under an inducible promoter. After purification by nickel-chelate affinity chromatography, the recombinant product was detected as a 47-kDa protein band in immunoblots with the anti-ScMp65 serum, as well as with CaMp65-specific monoclonal antibodies. Both ScMp65 and CaMp65 were assayed for antigenic stimulation in cultures of peripheral blood mononuclear cells (PBMC) from 10 unselected human donors. While ScMp65 was substantially nonstimulatory, both rCaMp65 and the native CaMp65 were equally able to induce lymphoproliferation of the PBMC from all the donors. In addition, a number of CD4+ T-cell clones were generated using a C. albicans mannoprotein fraction as an antigenic stimulant. Several of these clones specifically responded to both the native and the recombinant C. albicans Mp65 but not to ScMp65. Thus, the recombinant Mp65 of C. albicans retains antigenicity and, as such, could be a valid, standardized reagent for serodiagnostic and immunological studies.

Beneficial effect of Mentha suaveolens essential oil in the treatment of vaginal candidiasis assessed by real-time monitoring of infection

BackgroundVaginal candidiasis is a frequent and common distressing disease affecting up to 75% of the women of fertile age; most of these women have recurrent episodes. Essential oils from aromatic plants have been shown to have antimicrobial and antifungal activities. This study was aimed at assessing the anti-fungal activity of essential oil from Mentha suaveolens (EOMS) in an experimental infection of vaginal candidiasis.MethodsThe in vitro and in vivo activity of EOMS was assessed. The in vitro activity was evaluated under standard CLSI methods, and the in vivo analysis was carried out by exploiting a novel, non-invasive model of vaginal candidiasis in mice based on an in vivo imaging technique.Differences between essential oil treated and saline treated mice were evaluated by the non-parametric Mann-Whitney U-test. Viable count data from a time kill assay and yeast and hyphae survival test were compared using the Students t-test (two-tailed).ResultsOur main findings were: i) EOMS shows potent candidastatic and candidacidal activity in an in vitro experimental system; ii) EOMS gives a degree of protection against vaginal candidiasis in an in vivo experimental system.ConclusionsThis study shows for the first time that the essential oil of a Moroccan plant Mentha suaveolens is candidastatic and candidacidal in vitro, and has a degree of anticandidal activity in a model of vaginal infection, as demonstrated in an in vivo monitoring imaging system. We conclude that our findings lay the ground for further, more extensive investigations to identify the active EOMS component(s), promising in the therapeutically problematic setting of chronic vaginal candidiasis in humans.

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.

Modular structure of HEL protein from Arabidopsis reveals new potential functions for PR-4 proteins.

Abstract Plants possess an innate immune system enabling them to defend themselves against pathogen attack. The accumulation of newly synthesized pathogenesis-related proteins (PRs) is one of the most studied inducible plant defence response. In this paper, we report on the characterization of a class I PR4 vacuolar protein from Arabidopsis, named AtHEL. The protein has a modular structure consisting of an N-terminal hevein-like domain (CB-HEL) and a C-terminal domain (CD-HEL) that are posttranslationally processed. Both domains show a strong antifungal activity, but they do not have chitinolitic properties. CD-HEL was found to be endowed with RNase, but not DNase activity. Molecular modeling carried out on both domains revealed that CB-HEL possesses a chitin binding site strictly conserved between hevein-type peptides and that the cavity involved in substrate interaction of CD-HEL do not show any residue substitution with respect to the orthologous wheatwin1 from wheat. Using a fishing for partners approach, CB-HEL was found to interact with a fungal fruiting body lectin. According to literature, we can hypothesize that CB-HEL could cross the pathogen hyphal membrane and that its interaction with a fungal lectin could knock out one of the weapons that the fungus uses.

Clinical and mycological evaluation of fluconazole in the secondary prophylaxis of esophageal candidiasis in AIDS patients. An open, multicenter study

A prospective, multicenter, open study of fluconazole prophylaxis was performed in AIDS patients to evaluate the efficacy and toxicity of the drug in preventing relapses of esophageal candidiasis. To this aim, 99 AIDS patients who presented a first episode of clinically and microbiologically confirmed esophageal candidiasis were enrolled in eleven clinical centers scattered throughout the Italian territory. After resolution of this initial esophagitis, all subjects were given fluconazole, 100 mg/die, and followed up for a 6 month period. Only 7 out of the 99 patients enrolled had a relapse ofCandida esophagitis, during a mean follow-up period of 138.5 days. All relapsing patients had CD4+ cell number <100/µl at baseline. Mild side effects were reported in only eight patients. However, 14 of the 27 subjects from whom serial serum samples were available became (12) or remained (2) antigenemic during fluconazole prophylaxis, independently from relapse, suggesting the persistence of tissue-invasive, proliferatingCandida cells. Overall, the data of this study suggest a beneficial effect of prophylactic maintenance therapy with fluconazole againstCandida esophagitis, particularly in the population with >100 CD4+/µl. However, the data onCandida antigenemia in these patients invite the consideration of a relative inefficiency of the drug to eradicate the microrganism from the esophageal tissue.