Detecção e quantificação da expressão do gene ERG11 de Candida albicans sob diferentes concentrações de fluconazol

Abstract

Candida species inhabit the human body, don’t cause diseases in organisms considered healthy, however can lead to serious infections in those with changes in the defense mechanisms and / or impairment of anatomical barriers. The most common etiological agent of infections caused by Candida spp. is C. albicans, including in Brazil. Many years ago, antifungal drugs of the azole class have been prescribed for treatments of such infections, however the incidence of cases of resistance has been documented. Overexpression of the ERG11 gene and efflux pumps and mutations in the ERG11 gene are described as molecular mechanisms of resistance to azoles in Candida spp. In this way, the analysis of the ERG11 gene expression of clinical isolates and sources of environmental colonization was the proposal of this study, involving treatments under fluconazole inhibitory and subinhibitory concentrations (FLU), never researched before. Thus, three isolates from hospital environment colonization sources and five clinical isolates were used, in addition to the standard strain ATCC 10231 whose minimum inhibitory concentrations for fluconazole were obtained according to the EUCAST Protocol De fi nitive Document EDef 7.1 (2008). The treatments consisted of minimal inhibitory concentrations (MIC) and subinhibitory equivalent to 1/4 and 1/2 of the CIM of antifungal and in the absence of the same. Quantifications of the RT-qPCR expressions were performed for the ERG11 genes, and the Actin normalizer. Relative expression analyzes were calculated using the 2-ΔΔCt method. It was observed the behavior of all the isolates analyzed, independent of the origin of isolation, including the standard strain ATCC 10231, significant increase of ERG11 gene expression in relation to treatments with MIC, 1/2 and 1/4 FLU MIC when compared to the same isolates in the absence of FLU. This increase ranged from 1,086 to 126,105. In the group of clinical isolates, the one that most expressed the target gene was 220 (126.10 times) and in the environmental colonization group was the 521 isolate (40,79), both in the treatment of MIC of fluconazole. In general, the highest dose of fluconazole (MIC) was the one that most influenced the isolates expressing ERG11, followed by treatments with 1/2 and 1/4 MIC. These results of increased expression of clinical isolates of C. albicans are relevant, especially in cases of patients in prophylactic treatment, when antifungal rather than protecting the patient from infection could be stimulating the pathogen. The fact of the environmental colonization isolates had presented the same behavior, it generates an alert for the importance in hygiene in the hospital environment and antisepsis of health professionals.