Caracterização de isolados de Candida spp de cavidade bucal quanto aos aspectos fenótípicos e moleculares e obtenção de mutantes heteroresistentes à antotericina B e fluconazol

Abstract

In the last years the increase in the incidence of superficial and invasive candidosis caused by resistant species has been attributed to the use of antibiotics and immunosuppression drugs. The increase do Candida non-albicans isolate due to the increment of patient immunocompromised, immunodeficiency virus infection, neutropenic, newly born of low weight, procedures invasives and iatrogenic. All those factors allow that agents a little virulent, caused diseases. Several biochemical and molecular studies have been accomplished for elucidating the causes of resistance in yeasts. The aims of the present research were: to study yeasts of the gender Candida isolated of the buccal cavity patients with and without HIV; comparison of methods of identification of isolates ; to determine the sensibility profile to the amphotericin B and fluconazole antifungals by microdilution methodologies CLSI, AFST-EUCAST and Etest difusion agar and acquisition of heteroresistent samples to amphotericin B and to fluconazole by induction and selection experiments. Were identifications 22 C. albicans, 3 C. tropicalis, 3 C. parapsilosis and 2 C. glabrata through the classic methods. The identification by morphologic and biochemisth was 100% agreement with the results obtained by the molecular method (PCR), them by the amplification of the species with specific primers (C. albicans and C. dubliniensis) as for the comparison of the size of the fragment amplified by the primers of region ITS2. The correlation among the methodology Etest and the two microdilution methods (CLSI and AFST-EUCAST) was higth. All samples wilds were suscetibility to amphotericin B (CIM£2,0μg/mL) and just one isolate (3,33%) of C. tropicalis was resistent to fluconazole (CIM³64,0μg/mL por CLSI e CIM³ 64,0μg/mL por AFST-EUCAST). Resistant strains were obtained by selective pressure using a medium amended with Fluconazole (8 μg/mL) and after that a medium with Amphothericin B (1.0 μg/mL). The selective pressure was also performed using Amphothericin B at first followed by Fluconazole. Similarly, wild samples were submitted to the induction of resistance by culturing in increasing concentrations of Fluconazole (16.0; 32.0; 64.0; 128 e 256mg/mL) followed by culturing in increasing concentrations of Amphotericin B (0.5; 1.0; 1.5; 2.0; 2.5 e 3.0mg/mL) and, after that, inverting the antifungal agents: Amphotericin B at first, followed by Fluconazole. Three samples heteroresistant to Fluconazole were obtained, two of which obtained by induction (C. glabrata and C. tropicalis) and one by selection (C. tropicalis.). Both C. tropicalis samples were originated from te same wild strain.None sample chamged its susceptibility profile to amphotericin B, in spite of some isolates that presented capacity of growth in several concentrations of this antifungals (1,0 a 3,0μg/mL). After successive cultivations in medium free of drugs, only the sample obtained by selection maintained your resistance phenotype. The wild samples and their mutants presented the same profile when compared by the amplification of the region ITS2. In conclusion, although most of the studied species were C. albicans (73,33%), all the fluconazol-heteroresistant samples obtained were Candida no-albicans (10%). The two mutant samples obtained from the same initial isolate possibly possess multiple and different mechanisms of resistance that, associated to the isolate, confer resistance and/or adaptability to the yeast in front to antifungal.