Maria Aparecida de Resende

Yeasts associated with fresh and frozen pulps of Brazilian tropical fruits.

The occurrence of yeasts on ripe fruits and frozen pulps of pitanga (Eugenia uniflora L), mangaba (Hancornia speciosa Gom.), umbu (Spondias tuberosa Avr. Cam.), and acerola (Malpighia glaba L) was verified. The incidence of proteolytic, pectinolytic, and mycocinogenic yeasts on these communities was also determined. A total of 480 colonies was isolated and grouped in 405 different strains. These corresponded to 42 ascomycetous and 28 basidiomycetous species. Candida sorbosivorans, Pseudozyma antarctica, C. spandovensis-like, C. spandovensis, Kloeckera apis, C. parapsilosis, Rhodotorula graminis, Kluyveromyces marxianus, Cryptococcus laurentii, Metchnikowia sp (isolated only from pitanga ripe fruits), Issatchenkia occidentalis and C. krusei (isolated only from mangaba frozen pulps), were the most frequent species. The yeast communities from pitanga ripe fruits exhibited the highest frequency of species, followed by communities from acerola ripe fruits and mangaba frozen pulps. Yeast communities from frozen pulp and ripe fruits of umbu had the lowest number of species. Except the yeasts from pitanga, yeast communities from frozen pulp exhibited higher number of yeasts than ripe fruit communities. Mycocinogenic yeasts were found in all of the substrates studied except in communities from umbu ripe fruits and pitanga frozen pulps. Most of the yeasts found to produce mycocins were basidiomycetes and included P. antarctica, Cryptococcus albidus, C. bhutanensis-like, R. graminis and R. mucilaginosa-like from pitanga ripe fruits as well as black yeasts from pitanga and acerola ripe fruits. The umbu frozen pulps community had the highest frequency of proteolytic species. Yeasts able to hydrolyse casein at pH 5.0 represented 38.5% of the species isolated. Thirty-seven percent of yeast isolates were able to hydrolyse casein at pH 7.0. Pectinolytic yeasts were found in all of the communities studied, excepted for those of umbu frozen pulps. The highest frequency of pectinolytic activity was found in mangaba frozen pulp communities. Around 30% of all isolates produced pectinases. The ability to split arbutin was observed in all communities ranging from 8% in yeasts from pitanga frozen pulps to 40.6% in acerola ripe fruit communities. Among 432 species tested, 125 were active for beta-glucosidase production, and Kloeckera apis, P. antarctica, C. sorbosivorans, and C. spandovensis-like were the most active species.

Antifungal properties of plants used in Brazilian traditional medicine against clinically relevant fungal pathogens

Antifungal properties of extracts from eight Brazilian plants traditionally used in popular Brazilian medicine were tested against five clinically relevant Candida species, Cryptococcus neoformans, and Sporothrix schenckii. Results demonstrate that almost all extracts exhibited antifungal activity, at least against one of the microorganisms tested. The ethanolic extract from the leaves of Schinus terebinthifolius exhibited potential antifungal activity against C. glabrata and S. schenckii. Preliminary phytochemical analysis of extract from S. terebinthifolius showed the presence of biologically active compounds, namely saponins, flavonoids, triterpenes, steroids and tannins.

In vitro photodynamic inactivation of Candida spp. growth and adhesion to buccal epithelial cells.

In this study, photodynamic inactivation (PDI) was used to inhibit in vitro growth and adhesion of different Candida isolates to buccal epithelial cells (BEC). Experimental conditions were optimized and 25muM toluidine blue O (TBO) and 15min of irradiation time by light emitting diode (LED) (energy density of 180J/cm(2)) were selected due to higher reductions in cellular viability obtained after treatment. Reduction media of Log(10) 3.41 in viable cellular growth and media of 55% in the inhibition of adhesion to buccal epithelial cells were obtained. Two fluconazole resistant isolates were susceptible to PDI (Log(10) 3.54 in IB05 and Log(10) 1.95 in CG09) and a second session of this treatment for CG09 isolate inhibited cellular viability in 100%, without producing heat. The results permit to conclude that photodynamic inactivation under these experimental conditions would be a possible alternative approach to inhibit Candida spp. cellular growth and adhesion to buccal epithelial cells.

Antifungal activity of extracts of some plants used in Brazilian traditional medicine against the pathogenic fungus Paracoccidioides brasiliensis

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by Paracoccidioides brasiliensis Almeida (Onygenales) that requires 1–2 years of treatment. In the absence of drug therapy, the disease is usually fatal, highlighting the need for the identification of safer, novel, and more effective antifungal compounds. With this need in mind, several plants employed in Brazilian traditional medicine were assayed on P. brasiliensis and murine macrophages. Extracts were prepared from 10 plant species: Inga spp. Mill. (Leguminosae), Schinus terebinthifolius Raddi (Anacardiaceae), Punica granatum L. (Punicaceae), Alternanthera brasiliana Kuntze (Amaranthaceae), Piper regnellii CDC. (Piperaceae), P. abutiloides Kunth (Piperaceae), Herissantia crispa L. Briz. (Malvaceae), Rubus urticaefolius Poir (Rosaceae), Rumex acetosa L. (Polygonaceae), and Baccharis dracunculifolia DC. (Asteraceae). Hexane fractions from hydroalcoholic extracts of Piper regnellii and Baccharis dracunculifolia were the most active against the fungus, displaying minimum inhibitory concentration (MIC) values of 7.8 μg/mL and 7.8–30 μg/mL, respectively. Additionally, neither of the extracts exhibited any apparent cytotoxic effects on murine macrophages at 20 μg/mL. Analyses of these fractions using gas chromatography-mass spectrometry (GC-MS) showed that the major components of B. dracunculifolia were ethyl hydrocinnamate (14.35%) and spathulenol (16.02%), while the major components of the hexane fraction of Piper regnellii were 1-methoxy-4-(1-propenyl) benzene (21.94%) and apiol (21.29%). The activities of these fractions against P. brasiliensis without evidence of cytotoxicity to macrophages justify their investigation as a potential source of new chemical agents for the treatment of PCM.

Photodynamic therapy for pathogenic fungi.

Photodynamic therapy (PDT) is a minimally invasive approach, in which a photosensitiser compound is activated by exposure to visible light. The activation of the sensitiser drug results in several chemical reactions, such as the production of oxygen reactive species and other reactive molecules, whose presence in the biological site leads to the damage of target cells. Although PDT has been primarily developed to combat cancerous lesions, this therapy can be employed for the treatment of several conditions, including infectious diseases. A wide range of microorganisms, including Gram positive and Gram negative bacteria, viruses, protozoa and fungi have demonstrated susceptibility to antimicrobial photodynamic therapy. This treatment might consist of an alternative to the management of fungal infections. Antifungal photodynamic therapy has been successfully employed against Candida albicans and other Candida species and also against dermatophytes. The strain‐dependent antifungal effect and the influence of the biological medium are important issues to be considered. Besides, the choice of photosensitiser to be employed in PDT should consider the characteristics of the fungi and the medium to be treated, as well as the depth of penetration of light into the skin. In the present review, the state‐of‐the‐art of antifungal PDT is discussed and the photosensitiser characteristics are analysed.

Photodynamic Antifungal Therapy Against Chromoblastomycosis

Photodynamic therapy (PDT) is a minimally invasive approach, in which a photosensitizer compound is activated by exposure to light. The activation of the sensitizer drug results in several chemical reactions, such as the production of reactive oxygen species and other reactive molecules, which presence in the biological site leads to the damage of target cells. Although PDT has been primarily developed to combat cancerous lesions, this therapy can be employed for the treatment of several conditions, including infectious diseases. A wide range of microorganisms, including Gram-positive and Gram-negative bacteria, viruses, protozoa, and fungi, have demonstrated susceptibility to antimicrobial PDT. This treatment might consist in an alternative for the management of fungal infections. Antifungal photodynamic therapy has been successfully employed against Candida species, dermatophytes, and Aspergillus niger. Chromoblastomycosis is an infection that involves skin and subcutaneous tissues caused by the traumatic inoculation of dematiaceous fungi species, being that the most prevalent are Fonsecaea pedrosoi and Claphialophora carrionii. In the present work, the clinical applications of PDT for the treatment of chromoblastomycosis are evaluated. We have employed methylene blue as photosensitizer and a LED (Light Emitting Diode) device as light source. The results of this treatment are positive, denoting the efficacy of PDT against chromoblastomycosis. Considering that great part of the published works are focused on in vitro trials, these clinical tests can be considered a relevant source of information about antifungal PDT, since its results have demonstrated to be promising. The perspectives of this kind of treatment are analyzed in agreement with the recent literature involving antifungal PDT.

Virulence and molecular diversity in Colletotrichum graminicola from Brazil.

Genetic diversity among 37 isolates of the sorghum anthracnose pathogen Colletotrichum graminicola, from four geographically distinct regions of Brazil, was evaluated by RAPD and RFLP-PCR markers and virulence characters on a set of 10 differential sorghum genotypes. Twenty-two races were identified and race 13B was the most frequent, but present in only two regions. RAPD analysis revealed 143 polymorphic bands that grouped the isolates according to their geographic origin, but not by their virulence phenotypes. RFLP with HaeIII, MspI, HinfI, HhaI, HpaII, EcoRI, HindIII, PstI, RsaI, Taq αI, and AluI enzymes over ITS domains and 5.8 rDNA genes of C. graminicola did not show differences among the isolates, indicating high conservation of these restriction sites. Molecular polymorphism was observed among isolates belonging to the same race. No association between virulence phenotypes and molecular profiles was observed.

Molecular Model for Studying the Uncultivated Fungal Pathogen Lacazia loboi

ABSTRACT Lacazia loboi is an uncultivated fungal pathogen of humans and dolphins that causes cutaneous and subcutaneous infections only in the tropical areas of the Americas. It was recently found by phylogenetic analysis that this unusual pathogen is closely related to Paracoccidioides brasiliensis and to the other fungal dimorphic members of the order Onygenales. That original phylogenetic study used universal primers to amplify well-known genes. However, this approach cannot be applied to the study of other proteins. We have developed a strategy for studying the gene encoding the gp43 homologous protein of P. brasiliensis in L. loboi. The gp43 protein was selected because it has been found that this P. brasiliensis antigen strongly reacts when it is used to test sera from patients with lacaziosis. The principle behind this idea was to obtain the gp43 amino acid sequence of P. brasiliensis and other homologous fungal sequences from GenBank and design primers from their aligned conserved regions. These sets of primers were used to amplify the selected regions with genomic DNA extracted from the yeast-like cells of L. loboi from experimentally infected mice. Using this approach, we amplified 483 bp of the L. loboi gp43-like gene. These sequences had 85% identity at the nucleotide level and 75% identity with the deduced amino acid sequences of the P. brasiliensis gp43 protein. The identity of the 483-bp DNA fragment was confirmed by phylogenetic analysis. This analysis revealed that the L. loboi gp43-like deduced amino acid sequence formed a strongly supported (100%) sister group with several P. brasiliensis gp43 sequences and that this taxon in turn was linked to the other fungal sequences used in this analysis. This study shows that the use of a molecular model for investigation of the genes encoding important proteins in L. loboi is feasible.

Six years' experience in treatment of chromomycosis with 5-fluorocytosine.

Twenty‐three patients with chromomycosis were treated with oral 5‐fluorocytosine for periods ranging from 2 to 67 months. Sixteen were considered cured, after 3 consecutive monthly controls showing healing of the lesions and absence of fungi in mycologic and histopathologic examinations of biopsy specimens. Seven, in spite of initial clinical improvement, showed resistance to treatment. Associated treatment with amphotericin B, calciferol or thiabendazole failed to help patients resistant to 5‐fluorocytosine. Resistance seems to occur especially in those with long‐standing lesions or widespread involvement.

Antifungal activity of the amyrin derivatives and in vitro inhibition of Candida albicans adhesion to human epithelial cells.

Aims:  The antifungal activity of amyrin pentacyclic triterpene and 15 synthetic derivatives was evaluated against Candida species. Additionally, inhibition of adhesion of Candida albicans to human epithelial cells in vitro was determined.