Effects of Amphotericin B and Three Azole Derivatives on the Lipids of Yeast Cells of Paracoccidioides brasiliensis

Abstract

Yeast cells of five different strains of Paracoccidioides brasiliensis were obtained for partial analysis of lipid composition, and sterol content was determined quantitatively and qualitatively. The determinations were conducted with cells cultured in the presence and absence of amphotericin B and azole derivatives at levels below the MIC.Paracoccidioidomycosis is a chronic granulomatous disease with a diversity of clinical manifestations, and special emphasis has been placed on the pulmonary and mucocutaneous forms (29,42). The disease is limited to Latin American countries, and it is considered the most prevalent systemic mycosis in Latin America (27)(28)(29)38).The dimorphic fungus Paracoccidioides brasiliensis is the single etiologic agent of paracoccidioidomycosis, which is acquired by inhalation of spores of the mycelial phase of the fungus (3,38,43,45).In the present study we assessed the effects of amphotericin B, ketoconazole, itraconazole, and fluconazole on the sterols of five different P. brasiliensis strains in order to better evaluate the in vitro efficacy of these drugs. The MIC of each drug and the effects of the compounds on total sterol content were determined.Fungal strains and culture conditions. Five P. brasiliensis strains were studied. Of these, JT-1, a human isolate, corresponded to the reference strain ATCC 90659. Three other strains, called Pinguim, SN, and 18, were from the fungal collection of the Faculty of Medicine of the Universidade de São Paulo and were a kind gift from C. Fava-Netto (Universidade de São Paulo) and Patrícia Cisalpino (Instituto de Ciencias Biológicas, Universidade Federal de Minas Gerais). The environmental strain Pinguim was isolated from the feces of a penguin in the Antarctic and characterized as P. brasiliensis in mycologic and immunochemical studies (15). The human isolate RAJ-2 was a kind gift from Tomoko Tadano and originated from the Clinical Pathology Laboratory of the Julio Müller University Hospital, Cuiabá-MT, where it had been obtained from the sputum of a patient at the Julio Müller University Hospital.All strains were maintained on solid Fava-Netto medium at 22 to 28°C with monthly subculture and converted to the yeastlike phase by incubation at 35°C. The full transition of the forms was confirmed by the observation of the macroscopic and microscopic aspects of the colonies obtained.For the study of lipid composition and of the effects of amphotericin B and the three azoles, the microorganisms were adapted to solid McVeigh Morton (MVM) medium, modified as described previously (39).Antifungal agents. All strains were tested in the presence of amphotericin B, ketoconazole, fluconazole, and itraconazole. Amphotericin B was obtained from Sigma Chemical Company in pure form and was stored at 4°C in a dark glass bottle. The azole derivatives ketoconazole and itraconazole were kindly provided by Janssen in the form of pure salts and were stored at room temperature.The studies involving fluconazole were carried out using a commercial preparation (Fluconal...

Evaluation of Broth Microdilution Antifungal Susceptibility Testing Conditions for Trichophyton rubrum

“…Between MVM and SDB, the first was the medium which was more similar in comparison to RPMI. Although MVM is a chemically defined medium (20) often used in MIC determination for Paracoccidioides brasiliensis (11) by the broth macrodilution method, MIC reading in microdilution plates is harder because of the transparency of this medium compared to the yellow color of the other media, which confuses during visualization. There is no report about the use of MVM medium in MIC determination for dermatophytic fungi.…”

Eficiência terapêutica das formulações lipídicas de anfotericina B

“…Em relação aos fungos filamentosos, a AB possui amplo espectro de ação, incluindo Aspergillus fumigatus (MIC 1,0-2,0 mg/mL), Aspergillus flavus (MIC 1,0 mg/mL) (Espinel-Ingroff et al, 1997;Arikan et al, 1999), Fusarium oxysporum (MIC 2,0 mg/mL), Fusarium solani (MIC 1,0-2,0 mg/mL), Rhizopus arrhizuz (MIC 0,2-0,5 mg/mL) (Espinel-Ingroff et al, 1997) e Paracoccidioides brasiliensis (1,0-0,25 mg/mL) (Hahn, Hamdan, 2000).…”

Antifungal and antioxidant activity of fatty acid methyl esters from vegetable oils

“…Exactly 75 μL of sample or standard (1, 10, 100, 250 and 500 μg/mL) was added to 150 μL of DPPH, and the microplate was then covered and left in the dark (once) at room temperature (25 °C); 80% methanol was used for baseline correction. Hahn and Hamdan (2000). The resulting suspensions were diluted in RPMI 1640 (1 : 10) to obtain the final inoculum with 1-5 × 10 5 yeasts/ mL (Cruz et al 2013).…”