Antifungal activity of 3,3'-dimethoxycurcumin (DMC) against dermatophytes and Candida species.

Dermatomycosis is an infection with global impacts caused especially by dermatophytes and Candida species. Current antifungal therapies involve drugs that face fungal resistance barriers. This clinical context emphasizes the need to discover new antifungal agents. Herein, the antifungal potential of 10 curcumin analogs was evaluated against four Candida and four dermatophyte species. The most active compound, 3,3'-dimethoxycurcumin, exhibited minimum inhibitory concentration values ranging from 1.9‒62.5 to 15.6‒62.5 µg ml-1 against dermatophytes and Candida species, respectively. According to the checkerboard method, the association between DMC and terbinafine demonstrated a synergistic effect against Trichophyton mentagrophytes and Epidermophyton floccosum. Ergosterol binding test indicated DMC forms a complex with ergosterol of Candida albicans, C. krusei, and C. tropicalis. However, results from the sorbitol protection assay indicated that DMC had no effect on the cell walls of Candida species. The in vivo toxicity, using Galleria mellonella larvae, indicated no toxic effect of DMC. Altogether, curcumin analog DMC was a promising antifungal agent with a promising ability to act against Candida and dermatophyte species.

A potent candicidal peptide designed based on an encrypted peptide from a proteinase inhibitor.

Antimicrobial peptides (AMP) represent an alternative in the treatment of fungal infections associated with countless deaths. Here, we report a new AMP, named KWI-19, which was designed based on a peptide encrypted in the sequence of an Inga laurina Kunitz-type inhibitor (ILTI). KWI-19 inhibited the growth of Candida species and acted as a fungicidal agent from 2.5 to 20 µmol L-1, also showing synergistic activity with amphotericin B. Kinetic assays showed that KWI-19 killed Candida tropicalis cells within 60 min. We also report the membrane-associated mechanisms of action of KWI-19 and its interaction with ergosterol. KWI-19 was also characterized as a potent antibiofilm peptide, with activity against C. tropicalis. Finally, non-toxicity was reported against Galleria mellonella larvae, thus strengthening the interest in all the bioactivities mentioned above. This study extends our knowledge on how AMPs can be engineered from peptides encrypted in larger proteins and their potential as candicidal agents.

Unveiling the functional significance of the 14.3.3 protein: A key player in Paracoccidioides brasiliensis biofilm formation.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. The interaction mediated by the presence of adhesins on the fungal surface and receptors in the extracellular matrix of the host, as well as the biofilm formation, is essential in its pathogenesis. Adhesins such as gp43, enolase, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and 14-3-3 have been demonstrated in the Paracoccidioides brasiliensis (Pb18) strain and recognized as necessary in the fungus-host interaction. The Pb 18 strain silenced to 14-3-3 showed changes in morphology, virulence, and adhesion capacity. The study aimed to evaluate the role of adhesin 14-3-3 in P. brasiliensis biofilm formation and the differential expression of genes related to adhesins, comparing planktonic and biofilm forms. The presence of biofilm was also verified in sutures in vitro and in vivo. The silenced strain (Pb14-3-3 aRNA) was compared with the wild type Pb18, determining the differential metabolic activity between the strains by the XTT reduction assay; the biomass by violet crystal and the polysaccharides by safranin, even as morphological differences by microscopic techniques. Differential gene expression for adhesins was also analyzed, comparing the relative expression of these in planktonic and biofilm forms at different times. The results suggested that the silencing of 14-3-3 protein altered the ability to form biofilm and its metabolism. The quantity of biomass was similar in both strains; however, the formation of exopolymeric substances and polysaccharide material was lower in the silenced strain. Our results showed increased expression of enolase, GAPDH, and 14-3-3 genes in the first periods of biofilm formation in the Pb18 strain. In contrast, the silenced strain showed a lower expression of these genes, indicating that gene silencing can influence the expression of other genes and be involved in the biofilm formation of P. brasiliensis. In vitro and in vivo assays using sutures confirmed this yeast's ability to form biofilm and may be implicated in the pathogenesis of paracoccidioidomycosis.

Computer-made peptide RQ18 acts as a dual antifungal and antibiofilm peptide though membrane-associated mechanisms of action.

The spread of fungi resistant to conventional drugs has become a threatening problem. In this context, antimicrobial peptides (AMPs) have been considered as one of the main alternatives for controlling fungal infections. Here, we report the antifungal and antibiofilm activity and some clues about peptide RQ18's mechanism of action against Candida and Cryptococcus. This peptide inhibited yeast growth from 2.5 µM and killed all Candida tropicalis cells within 2 h incubation. Moreover, it showed a synergistic effect with antifungal agent the amphotericin b. RQ18 reduced biofilm formation and promoted C. tropicalis mature biofilms eradication. RQ18's mechanism of action involves fungal cell membrane damage, which was confirmed by the results of RQ18 in the presence of free ergosterol in the medium and fluorescence microscopy by Sytox green. No toxic effects were observed in murine macrophage cell lines and Galleria mellonella larvae, suggesting fungal target selectivity. Therefore, peptide RQ18 represents a promising strategy as a dual antifungal and antibiofilm agent that contributes to infection control without damaging mammalian cells.

Antimicrobial Activity of Honey against Oral Microorganisms: Current Reality, Methodological Challenges and Solutions.

Honey has been shown to have antimicrobial activity against different microorganisms, but its effects on oral biofilms are largely unknown. In this review, we analyzed the currently available literature on the antimicrobial activity of honey against oral biofilms in order to determine its potential as a functional food in the treatment and/or prevention of oral diseases. Here, we compare studies reporting on the antimicrobial activity of honey against systemic and oral bacteria, discuss methodological strategies, and point out current gaps in the literature. To date, there are no consistent studies supporting the use of honey as a therapy for oral diseases of bacterial origin, but current evidence in the field is promising. The lack of studies examining the antibiofilm activity of honey against oral microorganisms reveals a need for additional research to better define aspects such as chemical composition, the mechanism(s) of action, and antimicrobial action.

Phenolic Profile and the Antioxidant, Anti-Inflammatory, and Antimicrobial Properties of Açaí (Euterpe oleracea) Meal: A Prospective Study.

The mechanical extraction of oils from Brazilian açaí (Euterpe oleracea Mart) produces significant amounts of a byproduct known as "meal", which is frequently discarded in the environment as waste material. Nevertheless, plant byproducts, especially those from oil extraction, may contain residual polyphenols in their composition and be a rich source of natural bioactive compounds. In this study, the phenolic composition and in vitro biological properties of a hydroethanolic açaí meal extract were elucidated. The major compounds tentatively identified in the extract by high-resolution mass spectrometry were anthocyanins, flavones, and flavonoids. Furthermore, rhamnocitrin is reported in an açaí byproduct for the first time. The extract showed reducing power and was effective in scavenging the ABTS radical cation (820.0 µmol Trolox equivalent∙g-1) and peroxyl radical (975.7 µmol Trolox equivalent∙g-1). NF-κB activation was inhibited at 10 or 100 µg∙mL-1 and TNF-α levels were reduced at 100 µg∙mL-1. However, the antibacterial effects against ESKAPE pathogens was not promising due to the high concentration needed (1250 or 2500 µg∙mL-1). These findings can be related to the diverse polyphenol-rich extract composition. To conclude, the polyphenol-rich extract obtained from açaí meal showed relevant biological activities that may have great applicability in the food and nutraceutical industries.

A new Kunitz trypsin inhibitor from Erythrina poeppigiana exhibits antimicrobial and antibiofilm properties against bacteria.

Erythrina poeppigiana belongs to Fabaceae family (subfamily Papillionoideae) and is commonly found in tropical and subtropical regions in Brazil. Herein, we described the purification and characterization of a new Kunitz-type inhibitor, obtained from E. poeppigiana seeds (EpTI). EpTI is composed by three isoforms of identical amino-terminal sequences with a molecular weight ranging from 17 to 20 kDa. The physicochemical features showed by EpTI are common to Kunitz inhibitors, including the dissociation constant (13.1 nM), stability against thermal (37-100 °C) and pH (2-10) ranging, and the presence of disulfide bonds stabilizing its reactive site. Furthermore, we investigated the antimicrobial, anti-adhesion, and anti-biofilm properties of EpTI against Gram-positive and negative bacteria. The inhibitor showed antimicrobial activity with a minimum inhibitory concentration (MIC, 5-10 µM) and minimum bactericidal concentration (MBC) of 10 µM for Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Staphylococcus aureus, and Staphylococcus haemolyticus. The combination of EpTI with ciprofloxacin showed a marked synergistic effect, reducing the antibiotic concentration by 150%. The increase in crystal violet uptake for S. aureus and K. pneumoniae strains was approximately 30% and 50%, respectively, suggesting that the bacteria plasma membrane is targeted by EpTI. Treatment with EpTI at 1x and 10 x MIC significantly reduced the biofilm formation and prompted the disruption of a mature biofilm. At MIC/2, EpTI decreased the bacterial adhesion to polystyrene surface within 2 h. Finally, EpTI showed low toxicity in animal model Galleria mellonella. Given its antimicrobial and anti-biofilm properties, the EpTI sequence might be used to design novel drug prototypes.

Gene expression of Paracoccidioides virulence factors after interaction with macrophages and fibroblasts.

BACKGROUND: Paracoccidioidomycosis (PCM) is a systemic mycosis with high prevalence in Latin America that is caused by thermodimorphic fungal species of the Paracoccidioides genus. OBJECTIVES: In this study, we used quantitative polymerase chain reaction (qPCR) to investigate the expression of genes related to the virulence of Paracoccidioides brasiliensis (Pb18) and P. lutzii (Pb01) strains in their mycelial (M) and yeast (Y) forms after contact with alveolar macrophages (AMJ2-C11 cell line) and fibroblasts (MRC-5 cell line). METHODS: The selected genes were those coding for 43 kDa glycoprotein (gp43), enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 14-3-3 protein (30 kDa), phospholipase, and aspartyl protease. FINDINGS: In the Pb18 M form, the aspartyl protease gene showed the highest expression among all genes tested, both before and after infection of host cells. In the Pb18 Y form after macrophage infection, the 14-3-3 gene showed the highest expression among all genes tested, followed by the phospholipase and gp43 genes, and their expression was 50-fold, 10-fold, and 6-fold higher, respectively, than that in the M form. After fibroblast infection with the Pb18 Y form, the 14-3-3 gene showed the highest expression, followed by the phospholipase and aspartyl protease genes, and their expression was 25-fold, 10-fold, and 10-fold higher, respectively, than that in the M form. Enolase and aspartyl protease genes were expressed upon infection of both cell lines. After macrophage infection with the Pb01 Y form, the 14-3-3 gene showed the highest expression, followed by the phospholipase and aspartyl protease genes, and their expression was 18-fold, 12.5-fold, and 6-fold higher, respectively, than that in the M form. MAIN CONCLUSIONS: In conclusion, the data show that the expression of the genes analysed may be upregulated upon fungus-host interaction. Therefore, these genes may be involved in the pathogenesis of paracoccidioidomycosis.

Differential miRNA Expression in Human Macrophage-Like Cells Infected with Histoplasma capsulatum Yeasts Cultured in Planktonic and Biofilm Forms.

Histoplasma capsulatum affects healthy and immunocompromised individuals, sometimes causing a severe disease. This fungus has two morphotypes, the mycelial (infective) and the yeast (parasitic) phases. MicroRNAs (miRNAs) are small RNAs involved in the regulation of several cellular processes, and their differential expression has been associated with many disease states. To investigate miRNA expression in host cells during H. capsulatum infection, we studied the changes in the miRNA profiles of differentiated human macrophages infected with yeasts from two fungal strains with different virulence, EH-315 (high virulence) and 60I (low virulence) grown in planktonic cultures, and EH-315 grown in biofilm form. MiRNA profiles were evaluated by means of reverse transcription-quantitative polymerase chain reaction using a commercial human miRNome panel. The target genes of the differentially expressed miRNAs and their corresponding signaling pathways were predicted using bioinformatics analyses. Here, we confirmed biofilm structures were present in the EH-315 culture whose conditions facilitated producing insoluble exopolysaccharide and intracellular polysaccharides. In infected macrophages, bioinformatics analyses revealed especially increased (hsa-miR-99b-3p) or decreased (hsa-miR-342-3p) miRNAs expression levels in response to infection with biofilms or both growth forms of H. capsulatum yeasts, respectively. The results of miRNAs suggested that infection by H. capsulatum can affect important biological pathways of the host cell, targeting two genes: one encoding a protein that is important in the cortical cytoskeleton; the other, a protein involved in the formation of stress granules. Expressed miRNAs in the host's response could be proposed as new therapeutic and/or diagnostic tools for histoplasmosis.

Effect of Cinnamomum verum leaf essential oil on virulence factors of Candida species and determination of the in-vivo toxicity with Galleria mellonella model.

BACKGROUND: Essential oils (EO) extracted from Cinnamomum verum has been used as an antimicrobial agents for centuries. The effects of C. verum leaf oil against virulence of microorganisms is not well studied yet. OBJECTIVES: This study evaluates the effect of C. verum leaf oil against three virulence factors of Candida albicans, C. tropicalis and C. dubliniensis and its in-vivo toxicity. METHODS: Chemical composition of EO was determined using gas chromatography-mass spectrometry (GC-MS). Minimum inhibitory concentration (MIC) was determined using clinical and laboratory standards institute (CLSI) M27-A3 broth microdilution. Effect of EO on initial adhesion was quantified using XTT assay after allowing Candida cells to adhere to the polystyrene surface for 2 h. Biofilm formation of Candida in the presence of EO was quantified using XTT viability assay. Efficacy on reduction of germ tube formation was evaluated using standard protocol. Visualisation of biofilm formation and progression under the EO treatment were done using scanning electron microscope (SEM) and Time lapses microscope respectively. In-vivo toxicity of EO was determined using Galleria mellonella larvae. Chlorhexidine digluconate: positive control. RESULTS: Eugenol was the main compound of EO. MIC was 1.0 mg/mL. 50% reduction in initial adhesion was achieved by C. albicans, C. tropicalis and C. dubliniensis with 1.0, > 2.0 and 0.34 mg/mL respectively. 0.5 and 1.0 mg/mL significantly inhibit the germ tube formation. MBIC50 for forming biofilms were ≤ 0.35 mg/mL. 1.0 mg/mL prevent biofilm progression of Candida. SEM images exhibited cell wall damages, cellular shrinkages and decreased hyphal formation. No lethal effect was noted with in-vivo experiment model at any concentration tested. CONCLUSION: C. verum leaf oil acts against virulence factors of Candida and does not show any toxicity.

Phenolic profile and potential beneficial effects of underutilized Brazilian native fruits on scavenging of ROS and RNS and anti-inflammatory and antimicrobial properties.

Brazilian native fruits (BNF) have aroused interest of researchers and consumers for their great human health benefits. In this study, five BNF (Byrsonima lancifolia, Campomanesia phaea, Jacaratia spinosa, Solanum alternatopinnatum and Acnistus arborescens) were tested for their polyphenolic compounds by LC-ESI-MS/MS, reactive species deactivation (ROO˙, O2˙-, HOCl and NO˙), anti-inflammatory properties in vivo, and in vitro antimicrobial activity - with determination of putative mechanism(s) of action. Eighty-one polyphenols were identified, which exhibited a significant capacity to deactivate both ROS and RNS. C. phaea extract had the highest capacity to scavenge ROO˙ (68.94 µmol TE per g), O2˙- (IC50: 575.36 µg mL-1) and NO˙ (IC50: 16.96 µg mL-1), which may be attributed to the presence of ellagitanins. B. lancifolia decreased neutrophil influx into the peritoneal cavity of mice by 50% as compared to carrageenan and reduced Candida albicans biofilm viability by 3 log10 possibly due to complexation with cell membrane ergosterol. In summary, the BNF presented herein are good sources of bioactive compounds with positive effects on deactivation of biological reactive species, as well as with anti-inflammatory and antimicrobial activities, which can be altogether highly beneficial to human health.

Development of a novel anti-biofilm peptide derived from profilin of Spodoptera frugiperda.

Candida yeast infections are the fourth leading cause of death worldwide. Peptides with antimicrobial activity are a promising alternative treatment for such infections. Here, the antifungal activity of a new antimicrobial peptide-PEP-IA18-was evaluated against Candida species. PEP-IA18 was designed from the primary sequence of profilin, a protein from Spodoptera frugiperda, and displayed potent activity against Candida albicans and Candida tropicalis, showing a minimum inhibitory concentration (MIC) of 2.5 µM. Furthermore, the mechanism of action of PEP-IA18 involved interaction with the cell membrane (ergosterol complexation). Treatment at MIC and/or 10 × MIC significantly reduced biofilm formation and viability. PEP-IA18 showed low toxicity toward human fibroblasts and only revealed hemolytic activity at high concentrations. Thus, PEP-IA18 exhibited antifungal and anti-biofilm properties with potential applicability in the treatment of infections caused by Candida species.

Antibiofilm efficacy of tea tree oil and of its main component terpinen-4-ol against Candida albicans.

Candida infection is an important cause of morbidity and mortality in immunocompromised patients. The increase in its incidence has been associated with resistance to antimicrobial therapy and biofilm formation. The aim of this study was to evaluate the efficacy of tea tree oil (TTO) and its main component - terpinen-4-ol - against resistant Candida albicans strains (genotypes A and B) identified by molecular typing and against C. albicans ATCC 90028 and SC 5314 reference strains in planktonic and biofilm cultures. The minimum inhibitory concentration, minimum fungicidal concentration, and rate of biofilm development were used to evaluate antifungal activity. Results were obtained from analysis of the biofilm using the cell proliferation assay 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and confocal laser scanning microscopy (CLSM). Terpinen-4-ol and TTO inhibited C. albicans growth. CLSM confirmed that 17.92 mg/mL of TTO and 8.86 mg/mL of terpinen-4-ol applied for 60 s (rinse simulation) interfered with biofilm formation. Hence, this in vitro study revealed that natural substances such as TTO and terpinen-4-ol present promising results for the treatment of oral candidiasis.

Anti-inflammatory and anti-Candida Effects of Brazilian Organic Propolis, a Promising Source of Bioactive Molecules and Functional Food.

Brazilian organic propolis (BOP) is an unexplored Brazilian propolis that is produced organically and certified according to international legislation. Our results showed that BOP has strong anti-inflammatory effects and acts by reducing nuclear factor κB activation, tumor necrosis factor α release, and neutrophil migration. In addition, BOP6 exhibited antifungal activity on planktonic and biofilm cultures of Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, and Candida parapsisolis and reduced in vitro yeast cell adhesion to human keratinocytes at sub-inhibitory concentrations. BOP demonstrated significantly low toxicity in Galleria melonella larvae at antifungal doses. Lastly, a chemical analysis revealed the presence of caffeoyltartaric acid, 3,4-dicaffeoylquinic acid, quercetin, and gibberellins A7, A9, and A20, which may be responsible for the biological properties observed. Thus, our data indicate that BOP is a promising source of anti-inflammatory and antifungal molecules that may be used as a functional food.

Antibiofilm efficacy of tea tree oil and of its main component terpinen-4-ol against Candida albicans

Abstract Candida infection is an important cause of morbidity and mortality in immunocompromised patients. The increase in its incidence has been associated with resistance to antimicrobial therapy and biofilm formation. The aim of this study was to evaluate the efficacy of tea tree oil (TTO) and its main component - terpinen-4-ol - against resistant Candida albicans strains (genotypes A and B) identified by molecular typing and against C. albicans ATCC 90028 and SC 5314 reference strains in planktonic and biofilm cultures. The minimum inhibitory concentration, minimum fungicidal concentration, and rate of biofilm development were used to evaluate antifungal activity. Results were obtained from analysis of the biofilm using the cell proliferation assay 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and confocal laser scanning microscopy (CLSM). Terpinen-4-ol and TTO inhibited C. albicans growth. CLSM confirmed that 17.92 mg/mL of TTO and 8.86 mg/mL of terpinen-4-ol applied for 60 s (rinse simulation) interfered with biofilm formation. Hence, this in vitro study revealed that natural substances such as TTO and terpinen-4-ol present promising results for the treatment of oral candidiasis.

Diacetylcurcumin: a new photosensitizer for antimicrobial photodynamic therapy in Streptococcus mutans biofilms.

This study evaluated the effect of antimicrobial photodynamic therapy (aPDT) on S. mutans using diacetylcurcumin (DAC) and verified DAC toxicity. In vitro, S. mutans biofilms were exposed to curcumin (CUR) and DAC and were light-irradiated. Biofilms were collected, plated and incubated for colony counts. DAC and CUR toxicity assays were conducted with Human Gingival Fibroblast cells (HGF). In vivo, G. mellonella larvae were injected with S. mutans and treated with DAC, CUR and aPDT. The hemolymph was plated and incubated for colony counts. Significant reductions were observed when DAC and CUR alone were used and when aPDT was applied. HGF assays demonstrated no differences in cell viability for most groups. DAC and CUR reduced the S. mutans load in G. mellonella larvae both alone and with aPDT. Systematic toxicity assays on G. mellonella demonstrated no effect of DAC and CUR or aPDT on the survival curve.

Antimicrobial activity of nitrochalcone and pentyl caffeate against hospital pathogens results in decreased microbial adhesion and biofilm formation.

The present study investigated the antimicrobial, anti-adhesion and anti-biofilm activity of the modified synthetic molecules nitrochalcone (NC-E05) and pentyl caffeate (C5) against microorganisms which have a high incidence in hospital-acquired infections. The compounds were further tested for their preliminary systemic toxicity in vivo. NC-E05 and C5 showed antimicrobial activity, with minimum inhibitory concentrations (MICs) ranging between 15.62 and 31.25 µg ml-1. Treatment with NC-E05 and C5 at 1 × MIC and/or 10 × MIC significantly reduced mono or mixed-species biofilm formation and viability. At MIC/2, the compounds decreased microbial adhesion to HaCaT keratinocytes from 1 to 3 h (p < 0.0001). In addition, NC-E05 and C5 demonstrated low toxicity in vivo in the Galleria mellonella model at anti-biofilm concentrations. Thus, the chemical modification of these molecules proved to be effective in the proposed anti-biofilm activity, opening opportunities for the development of new antimicrobials.

Anadenanthera Colubrina vell Brenan: anti-Candida and antibiofilm activities, toxicity and therapeutical action.

We evaluated the antifungal and antibiofilm potential of the hydroalcoholic extract of bark from Anadenanthera colubrina (vell.) Brenan, known as Angico, against Candida spp. Antifungal activity was evaluated using the microdilution technique through the Minimum Inhibitory and Fungicide Concentrations (MIC and MFC). The antibiofilm potential was tested in mature biofilms formed by Candida species and analyzed through the counting of CFU/mL and scanning electron micrograph (SEM). In vivo toxicity and therapeutic action was evaluated in the Galleria mellonella model. The treatment with the extract, in low doses, was able to reduce the growth of planktonic cells of Candida species. MIC values range between 19.5 and 39 µg/mL and MFC values range between 79 and 625 µg/mL. In addition was able to reduce the number of CFU/mL in biofilms and to cause structural alteration and cellular destruction, observed via SEM. A. colubrina showed low toxicity in the in vivo assay, having not affected the viability of the larvae at doses below 100mg/kg and high potential in the treatment of C. albicans infection. Considering its high antifungal potential, its low toxicity and potential to treatment of infections in in vivo model, A. colubrina extract is a strong candidate for development of a new agent for the treatment of oral candidiasis.

In silico approaches for screening molecular targets in Candida albicans: A proteomic insight into drug discovery and development.

Candida species are opportunistic pathogens which can cause conditions ranging from simple mucocutaneous infections to fungemia and death in immunosuppressed and hospitalized patients. Candida albicans is considered to be the species mostly associated with fungal infections in humans and, therefore, the mostly studied yeast. This microorganism has survival and virulence factors which, allied to a decreased host immunity response, make infection more difficult to control. Today, the current limited antifungal arsenal and a dramatic increase in fungal resistance have driven the need for the synthesis of drugs with novel mechanisms of action. However, the development of a new drug from discovery to marketing takes a long time and is highly costly. The objective of this review is to show that with advances in biotechnology and biofinformatics, in silico tools such as molecular docking can optimize such a timeline and reduce costs, while contributing to the design and development of targeted drugs. Here we highlight the most promising protein targets in Candida albicans for the development of drugs with new mechanisms of action.

Interactions between Terpinen-4-ol and Nystatin on biofilm of Candida albicans and Candida tropicalis.

The aim of this study was to evaluate the antifungal activity of Terpinen-4-ol associated with nystatin, on single and mixed species biofilms formed by Candida albicans and Candida tropicalis, as well as the effect of terpinen-4-ol on adhesion in oral cells and the enzymatic activity. The minimum inhibitory concentrations and minimum fungicide concentrations of terpinen-4-ol and nystatin on Candida albicans and Candida tropicalis were determined using the microdilution broth method, along with their synergistic activity ("checkerboard" method). Single and mixed species biofilms were prepared using the static microtiter plate model and quantified by colony forming units (CFU/mL). The effect of Terpinen-4-ol in adhesion of Candida albicans and Candida tropicalis in coculture with oral keratinocytes (NOK Si) was evaluated, as well as the enzymatic activity by measuring the size of the precipitation zone, after the growth agar to phospholipase, protease and hemolysin. Terpinen-4-ol (4.53 mg mL-1) and nystatin (0.008 mg mL-1) were able to inhibit biofilms growth, and a synergistic antifungal effect was showed with the drug association, reducing the inhibitory concentration of nystatin up to 8 times in single biofilm of Candida albicans, and 2 times in mixed species biofilm. A small decrease in the adhesion of Candida tropicalis in NOK Si cells was showed after treatment with terpinen-4-ol, and nystatin had a greater effect for both species. For enzymatic activity, the drugs showed no action. The effect potentiated by the combination of terpinen-4-ol and nystatin and the reduction of adhesion provide evidence of its potential as an anti-fungal agent.