Influência da exposição a antifúngicos clínicos na susceptibilidade, fisiologia, virulência e estresse oxidativo e nitrosativo em agentes causadores de dermatofitose

Abstract

The emergence of resistance to antifungals and the therapeutic failure resulting from this process is an increasingly recurrent fact in clinical practice. Resistance can be defined as a reduction in the effectiveness of a drug against an entire population of fungal cells. For dermatophytes, the resistance mechanisms are still not fully elucidated. Furthermore, the lower incidence of resistance to certain antifungal agents suggests the occurrence of mechanisms of action parallel to those described. Thus, this research evaluated the influence of exposure of dermatophytes to the antifungals terbinafine and itraconazole on fungal susceptibility, virulence and fungus-host interaction in vitro and in vivo, and analyzed the role of oxidative and nitrosative stress in treatment in vitro with amorolfine and ciclopirox olamine. In the first chapter, sixteen wild strains of dermatophytes underwent an adaptation process in medium supplemented with itraconazole or terbinafine and, later, cultivated in medium in the absence of these antifungals. Eleven strains (70.58%) were less susceptible to itraconazole and five (29.41%) to terbinafine. In addition, these strains also exhibited lower susceptibility to other clinical antifungals such as amorolfine, ketoconazole and ciclopirox olamine. Exposure to itraconazole increased the expression of the ERG11 gene in strains previously exposed to itraconazole. One strain also exhibited increased expression of the MDR3 gene. The decreased susceptibility and subculture in the absence of the antifungal caused changes in growth, conidia germination, tolerance to different sources of stress, biofilm production, interaction with macrophages and decreased fungal viability in a murine model. These results demonstrate that strains less susceptible to antifungals behave differently from strains that were not exposed to the drug, which results, in addition to changes in the susceptibility, physiology and expression of ERG11 and MDR3, in a differentiated interaction with macrophages and in a murine model. In the second chapter, three strains of dermatophytes were exposed to treatment with amorolfine and ciclopirox olamine to evaluate the production of reactive oxygen species (ROS) and peroxynitrite (PRN). These strains were also evaluated under treatment in the presence of ROS and PRN inhibitors: 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt monohydrate (Tiron) and 5,10,15,20-Tetrakis(4-trimethylammoniophenyl) porphyrin (FeTPPS). The three strains were susceptible to amorolfine and ciclopirox at 0.25 and 2.00 µg/mL, respectively. Treatment with amorolfine and ciclopirox increased ROS and PRN production and decreased post-treatment fungal burden. In the presence of inhibitors, the production of ROS and PRN decreased and the fungal burden increased. The combination of antifungals and inhibitors DETC, 3AT, Tiron, FeTPPS was also evaluated. The interactions were antagonistic for the combination of DETC with amorolfine and ciclopirox, synergistic for the combination of amorolfine to FeTPPS and 3AT, and indifferent for the other combinations. Treatment with amorolfine and ciclopirox reduced ergosterol content and increased lipid peroxidation. The association of amorolfine with inhibitors reduced lipid peroxidation and increased ergosterol content. This profile was also observed in the association of ciclopirox to FeTPPS, which indicates that amorolfine and ciclopirox have an antifungal action that also involves the production of ROS and PRN against T. interdigitale as a mechanism of action. Furthermore, this dataset demonstrates, first, the relative ease of production of strains with less susceptibility to antifungals, which makes this issue extremely worrisome and relevant. Second, a parallel mechanism of action for amorolfine and ciclopirox is established, which may explain the lower rate of resistance against these antifungals and establish paths for new therapeutic proposals.