The basidiomycete Cryptococcus neoformans is a pathogenic yeast responsible for causing meningoencephalitis in patients with a compromised immune system, leading to high mortality rates worldwide. Presently available antifungal drugs for treating this disease are not always efficient and they may lead to serious side effects and sometimes to fungal resistance. This situation has caused an increasing search for natural products derived from plants with antifungal action. Cymbopogon winterianus Jowitt (Poaceae), popularly known as citronella and Thymus vulgaris L. (Labiatae), known as thyme, are two plants commonly used in folk medicine. This study aimed at evaluating the biological activity of both C. winterianus and T. vulgaris essential oils (EOs) on C. neoformans clinical isolates. We evaluated the in vitro susceptibility of C. neoformans to each EO by the broth microdilution method, the in vitro interaction between EOs and fluconazole by the checkerboard method, their toxicity on human erythrocytes and their effects on the mitochondrial metabolism of fungal cells. Both EOs showed to possess antifungal activity against the tested isolates, with minimum inhibitory concentrations (MICs) ranging from 32 to 256 μg/mL for C. winterianus and from 32 to 128 μg/mL for T. vulgaris. No synergism between the EOs and fluconazole was observed. C. winterianus and T. vulgaris EOs did not cause hemolysis on human erythrocytes at concentrations corresponding to MIC values, indicating, therefore, low toxicity. The MTT assays showed no alterations on mitochondrial metabolism of fungal cells by C. winterianus at concentrations ≤ 128μg/mL, as well as no alterations at any concentrations corresponding to MIC values by T. vulgaris, suggesting that the main mechanism of action of both EOs is not by interfering with mitochondrial activity. Results show that C. winterianus and T. vulgaris EOs may be promising on the production of new antifungal drugs.

Vulvovaginal Candidiasis is the second most common infection in women of childbearing age, in about 70 to 90% of cases; the associated etiologic agent is C. albicans. The increase in the incidence of non-albicans species has led to the emergence of cases of resistance to antifungals, which is why the importance of new approaches in treatment. The objective of the present work was to develop and test in vivo a nanostructured system containing fluconazole and Green Propolis for the treatment of Vulvovaginal Candidiasis. The nanoparticles containing Fluconazole and Green Propolis were prepared following the technique of ionic gelation and presented satisfactory physical characteristics for the intended purpose. The association efficiency for the complexed compounds was performed using the technique of High Performance Liquid Chromatography (HPLC) and UV spectrophotometry. The Minimum Inhibitory Concentrations (MIC) for Fluconazole, Green Propolis and nanoparticles were determined, showing efficacy for use and fungistatic action on C. albicans ATCC 10231. Release analyzes of up to 72 hours with stabilization up to 96 hours were verified in the nanoparticles. The toxicity tests by hemolysis and Galleria mellonella, demonstrated positive results for propolis and negative for nanoparticles, suggesting safety in their use in a method with a murine model. In vivo tests were performed with an indication of efficacy for the proposed formulation.

O fluconazol é um fármaco antifúngico de amplo espectro muito utilizado pela população. Contudo, são poucos os registros na literatura que imputem a segurança do uso do fluconazol e tampouco a dose mínima capaz de induzir lesões no material genético do paciente. Embora a Agência Nacional de Vigilância Sanitária (ANVISA) exija testes de genotoxicidade para admissão de novos medicamentos, os que têm seu registro expedido antes do ano de 2006 não trazem essa obrigatoriedade, incluindo-se aqui o fluconazol. Portanto, é de extrema importância estudos sobre a avaliação genotoxicológica de fármacos, principalmente os que são tão conhecidos e utilizados como o fluconazol. O presente estudo investigou os efeitos genotóxicos da cápsula e do princípio ativo do fluconazol através do teste cometa e do teste de proliferação celular e também os efeitos citotóxicos da cápsula e do princípio ativo do fluconazol através do teste de viabilidade celular. As concentrações testadas da cápsula e do princípio ativo do fluconazol foram 6, 12, 30, 60 e 120 μg/mL. Todos os ensaios foram realizados em triplicatas e o peróxido foi utilizado como controle positivo e tampão PBS 7,4 como controle negativo. Todas as concentrações testadas da cápsula do Fluconazol (6, 12, 30, 60 e 120 μg/mL) demonstraram ser capazes de interferir negativamente no processo de proliferação celular, diminuindo o número de células proliferadas em todas as concentrações testadas, quando comparadas ao controle negativo. Já no caso do princípio ativo do Fluconazol, apenas as três últimas concentrações (30, 60 e 120 μg/mL) interferiram negativamente no processo de proliferação celular. Na avaliação do índice de dano ao DNA (teste cometa), foi observado dano à estrutura de DNA nas concentrações 60 e 120 μg/mL da cápsula do Fluconazol. Já as concentrações testadas com o princípio ativo do Fluconazol não foi observado dano à estrutura de DNA. Foi verificado que a cápsula do Fluconazol possui atividade citotóxica sobre leucócitos humanos nas duas maiores concentrações testadas (60 e 120 μg/mL) de forma concentração dependente. Porém, o princípio ativo do Fluconazol não demonstrou ser citotóxico nas concentrações testadas (6 12, 30, 60 e 120 μg/mL).

Candida glabrata has emerged as a common cause of serious life-threatening fungal infections, largely owing to their low susceptibility to azole antifungals. Recent guidance indicates the use of echinocandins as the first-choice drug for the treatment of systemic infections of C. glabrata; however, C. glabrata resistance to echinocandins is reportedly increasing. Herein, we present the induction of anidulafungin resistance in planktonic and sessile cells of C. glabrata and the development of fluconazole crossresistance. MICs of 21 clinical C. glabrata strains were determined by a broth microdilution method using anidulafungin and fluconazole. Biofilm formation on a tracheal catheter was determined using 1- × 1-cm2 polyvinyl polychloride catheter fragments. Induction of anidulafungin resistance in planktonic and sessile cells and evaluation of its stability were performed by exposing the strains to successively higher concentrations of the antifungal. The induction resulted in strains strongly resistant to anidulafungin (MICs: 1−2 μg/mL) and fluconazole (≥64 μg/mL). Most of the sessile cells of C. glabrata presented slightly reduced susceptibility compared with the planktonic cells. Clinically, this cross-resistance could lead to therapeutic failure while using fluconazole in patients previously exposed to subinhibitory concentrations of anidulafungin for extended periods.

Present study was aimed to prepare and characterize fluconazole loaded nanostructured lipid carriers (FLZ-NLCs) for the treatment of fungal infections. Fungal infections are tremendously widespread and are the often faced dermatological condition worldwide. FLZ-NLCs was prepared by ultrasonication emulsion technique using stearic acid (SA) as solid lipid, castor oil as liquid lipid and tween 20 as a surfactant. The mean diameter of optimized FLZ-NLCs were found to be 359.15 ± 9.83 nm. The drug content and entrapment efficiency of NLCs was found to be 102.97 ± 7.45% and 87 ± 0.59%, respectively. In vitro drug release studies of FLZ-NLCs showed 37.34 ± 2.08% drug release over a period of 72 h. The above studies confirmed the prepared FLZ-NLCs may be useful for the treatment of fungal infections.

The present study investigated the association of the non-steroidal anti-inflammatory drug ibuprofen with itraconazole, fluconazole and amphotericin B against Cryptococcus neoformans isolates. The minimal inhibitory concentration (MIC) was found according to M27-A3 protocol and in vitro interactions were evaluated using checkerboard microdilution method. Synergism was demonstrated between azoles and ibuprofen for most isolates. However, no synergistic effects were seen when amphotericin B was combined with ibuprofen. Therefore, our results suggest that ibuprofen presents clinical potential when combined with azole drugs in the treatment of cryptococcosis.

Candida glabrata infections are responsible for deaths of people globally. Fluconazole is known to be less effective against C. glabrata, which developed many strategies to evade being destroyed by fluconazole. To achieve enhanced efficacy of fluconazole against C. glabrata, the interaction of fluconazole with sodium bicarbonate was investigated using the CLSI guidelines. The efficacy of fluconazole alone and in combination with sodium bicarbonate was evaluated using the time-kill and phospholipase production assays. Eventually, the expression of PLB was assessed using semi-quantitative RT-PCR to investigate the inhibitory properties of fluconazole alone and in combination with sodium bicarbonate against C. glabrata. The fluconazole/sodium bicarbonate combination displayed synergistic and antagonistic effects (FICI= 0.375-4.25). In C. glabrata ATCC, SN 152, and SN 164, the fluconazole/sodium bicarbonate combination exhibited a significant fungicidal activity (p< 0.05) but antagonistic effect in the case of SN 283. With exception of SN 283, a significant reduction was noted in phospholipase production in clinical isolates of C. glabrata treated with fluconazole/sodium bicarbonate combination. The PLB was down-regulated significantly by 0.168-0.515 fold in C. glabrata treated with fluconazole/sodium bicarbonate. The results suggested fluconazole/sodium bicarbonate to have a potential synergistic interaction in C. glabrata, and the underlying mechanism may be associated with phospholipase gene.

Regulated erroneous protein translation (adaptive mistranslation) increases proteome diversity and produces advantageous phenotypic variability in the human pathogen Candida albicans. It also increases fitness in the presence of fluconazole, but the underlying molecular mechanism is not understood. To address this question, we evolved hypermistranslating and wild-type strains in the absence and presence of fluconazole and compared their fluconazole tolerance and resistance trajectories during evolution. The data show that mistranslation increases tolerance and accelerates the acquisition of resistance to fluconazole. Genome sequencing, array-based comparative genome analysis, and gene expression profiling revealed that during the course of evolution in fluconazole, the range of mutational and gene deregulation differences was distinctively different and broader in the hypermistranslating strain, including multiple chromosome duplications, partial chromosome deletions, and polyploidy. Especially, the increased accumulation of loss-of-heterozygosity events, aneuploidy, translational and cell surface modifications, and differences in drug efflux seem to mediate more rapid drug resistance acquisition under mistranslation. Our observations support a pivotal role for adaptive mistranslation in the evolution of drug resistance in C. albicans. IMPORTANCE Infectious diseases caused by drug-resistant fungi are an increasing threat to public health because of the high mortality rates and high costs associated with treatment. Thus, understanding of the molecular mechanisms of drug resistance is of crucial interest for the medical community. Here we investigated the role of regulated protein mistranslation, a characteristic mechanism used by C. albicans to diversify its proteome, in the evolution of fluconazole resistance. Such codon ambiguity is usually considered highly deleterious, yet recent studies found that mistranslation can boost adaptation in stressful environments. Our data reveal that CUG ambiguity diversifies the genome in multiple ways and that the full spectrum of drug resistance mechanisms in C. albicans goes beyond the traditional pathways that either regulate drug efflux or alter the interactions of drugs with their targets. The present work opens new avenues to understand the molecular and genetic basis of microbial drug resistance.

Nowadays, fungal infections are a serious public health issue due to the increasing drug resistance and small number of antifungals available. Candida albicans is the most prevalent human fungal pathogen, causing invasive fungal infections that are associated with high mortality rates. This fungus has a particular hybrid tRNA (tRNACAGSer) that is recognized by both the leucyl - and the seryl-tRNA synthetases (LeuRS and SerRS), allowing for incorporation of leucine (3%) and serine (97%) at CUG positions. It has already been shown that in the presence of antifungals, the level of Leu misincorporation increases up to 20% and that hypermistranslating strains have higher tolerance to drugs, namely azoles, which includes fluconazole. In this study, we tested the hypothesis that mistranslation could be directly linked with the appearance of drug tolerant subpopulations of C. albicans cells from which, with prolonged drug treatment, resistance may emerge. In order to understand if antifungals can select subpopulations of tolerant cells, we carried out in vitro competition experiments with fluorescently tagged C. albicans strains that were experimentally evolved with or without fluconazole during 400 generations. A wild-type strain (T0) was tagged with mCherry while hypermistranslating strains were tagged with GFP which allowed strain differentiation within the competition. Results showed an increase of hypermistranslating cells during evolution in the presence of the antifungal, but no significative alteration of the minimal inhibitory concentration (MIC) value was detected. On the other hand, microcolonies (constituted exclusively by hypermistranslator cells) appeared within the inhibition ellipse of the E test, suggesting the emergence of tolerance instead of resistance. These results suggest that prolonged antifungal therapy may select hypermistranslating clones that drive the appearance and persistence of tolerant cells within the population. Tolerance phenotypes may not be detected through significative MIC alterations but it must be taken into consideration since it could be associated with recurrent candidiasis