Photodynamic inactivation by hypericin-P123 on azole-resistant isolates of the Trichophyton rubrum complex as planktonic cells and biofilm.

INTRODUCTION: The Trichophyton rubrum complex comprises the majority of dermatophyte fungi (DM) responsible for chronic cases of onychomycosis, which is treated with oral or topical antifungals. However, owing to antifungal resistance, alternative therapies, such as photodynamic therapy (PDT), are needed. This study investigated the frequency of the T. rubrum species complex in onychomycosis cases in the northwestern region of Paraná state, Brazil, and evaluated the efficacy of (PDT) using P123-encapsulated hypericin (Hyp-P123) on clinical isolates of T. rubrum in the planktonic cell and biofilm forms. MATERIAL AND METHODS: The frequency of the T. rubrum complex in onychomycosis cases from 2017 to 2021 was evaluated through a data survey of records from the Laboratory of Medical Mycology (LEPAC) of the State University of Maringa (UEM). To determine the effect of PDT-Hyp-P123 on planktonic cells of T. rubrum isolates, 1 × 105 conidia/mL were treated with ten different concentrations of Hyp-P123 and then irradiated with 37.8 J/cm2. Antibiofilm activity of PDT-Hyp-P123 was tested against T. rubrum biofilm in the adhesion phase (3 h), evaluated 72 h after irradiation (37.8 J/cm2), and the mature biofilm (72 h), evaluated immediately after irradiation. In this context, three different parameters were evaluated: cell viability, metabolic activity and total biomass. RESULTS: The T. rubrum species complex was the most frequently isolated DM in onychomycosis cases (approximately 80 %). A significant reduction in fungal growth was observed for 75 % of the clinical isolates tested with a concentration from 0.19 µmol/L Hyp-P123, and 56.25 % had complete inhibition of fungal growth (fungicidal action); while all isolates were azole-resistant. The biofilm of T. rubrum isolates (TR0022 and TR0870) was inactivated in both the adhesion phase and the mature biofilm. CONCLUSION: PDT-Hyp-P123 had antifungal and antibiofilm activity on T. rubrum, which is an important dermatophyte responsible for onychomycosis cases.

Human Nails Permeation of an Antifungal Candidate Hydroalcoholic Extract from the Plant Sapindus saponaria L. Rich in Saponins.

We evaluated a hydroalcoholic extract of Sapindus saponaria L. pericarps (ETHOSS), as a candidate to a topical antifungal medicine for onychomycosis. ETHOSS was produced by extracting the crushed fruits in ethanol. The saponin contents were identified and characterized by electrospray ionization mass spectrometry. We measured the in vitro antifungal activity against three dermatophyte fungi, isolated from onychomycosis: Trichophyton rubrum, T. mentagrophytes, and T. interdigitale, using broth microdilution tests. The minimum fungicide concentration of ETHOSS ranged from 195.31 to 781.25 µg/mL. The cytotoxicity of the crude extract was tested on the HeLa cell line, and its ability to permeate into healthy human nails by photoacoustic spectroscopy and Fourier transformation infrared spectrometer (FTIR) spectroscopy by attenuated total reflection. Besides its strong antifungal activity, ETHOSS showed low cytotoxicity in human cells. It was able to permeate and reach the full thickness of the nail in one hour, without the aid of facilitating vehicles, and remained there for at least 24 h. These results suggest that ETHOSS has great potential for treating onychomycosis.

Preclinical approaches in vulvovaginal candidiasis treatment with mucoadhesive thermoresponsive systems containing propolis.

Vulvovaginal candidiasis (VVC) is a common vaginitis that affects women, especially in childbearing age, caused by Candida albicans in almost 80% of cases. Considering the limited drug arsenal available and the increasing fungal resistance profile, the search for new therapeutic sources with low toxicity and easy administration should be supported. Propolis has been used as a traditional medicine for multiple diseases, considering its particular composition and pharmaceutical properties that permits its wide applicability; it has also emerged as a potential antifungal agent. Thus, this study performed an in vitro and in vivo investigation into the efficacy of a new mucoadhesive thermoresponsive platform for propolis delivery (MTS-PRPe) in a preclinical murine model of VVC treatment caused by C. albicans. The methodologies involved chemical analysis, an assessment of the rheological and mucoadhesive properties of propolis formulations, in vitro and in vivo antifungal evaluations, histological evaluations and electron microscopy of the vaginal mucosa. The results demonstrated the antifungal activity of propolis extract and MTS-PRP against the standard strain and a fluconazole-resistant clinical isolate of C. albicans, in both in vitro and in vivo assays. These results were similar and even better, depending on the propolis concentration, when compared to nystatin. Thus, the formulation containing propolis exhibited good performance against C. albicans in a vulvovaginal candidiasis experimental model, representing a promising opportunity for the treatment of this infection.

In vitro antifungal activity and phytochemical characterization of Eugenia uniflora, Libidibia ferrea and Psidium guajava

The aim of this study was evaluate the in vitro antifungal activity of crude extracts from Eugenia uniflora, Libidibia ferrea and Psidium guajava. The extracts were obtained by turbo-extraction using water (AQ) or acetone-water (AC-W) (7:3, v/v) as solvents and lyophilized to obtain the crude extracts (CE). The CE were characterized by UV-Vis, TLC and HPLC. The activity of CEs was investigated against clinical isolates of Candida spp. and the Minimum Inhibitory Concentration (MIC), MIC50 and MIC90 were determinated. The analysis by TLC showed that all CEs presented polyphenols (flavonoids and tannins). The CEs from E. uniflora showed higher amount of polyphenols (30.35 ± 2.15%, AC-W) and the HPLC analysis revealed the tannins in all extracts. The CEs of E. uniflora showed MIC range from 1.9 to 500.0 µg/mL, and lower values of MIC50 and MIC90 against non-albicans Candida isolates. Regarding L. ferrea and P. guajava, the results showing MIC from 3.9 to 1000.0 µg/mL (CE-AQ) against C. albicans. The results demonstrate antifungal performance from CE against various species of Candida spp., suggesting that the herbal species may be use as new potential antifungal agents. Additionally, the polyphenol content can play a pivotal role in the antifungal properties of CE.

Antifungal activity of two oxadiazole compounds for the paracoccidioidomycosis treatment.

Paracoccidioidomycosis (PCM) is a neglected disease present in Latin America with difficulty in treatment and occurrence of serious sequelae. Thus, the development of alternative therapies is imperative. In the current work, two oxadiazole compounds (LMM5 and LMM11) presented fungicidal activity against Paracoccidioides spp. The minimum inhibitory and fungicidal concentration values ranged from 1 to 32 µg/mL, and a synergic effect was observed for both compounds when combined with Amphotericin B. LMM5 and LMM11 were able to reduce CFU counts (≥2 log10) on the 5th and 7th days of time-kill curve, respectively. The fungicide effect was confirmed by fluorescence microscopy (FUN-1/FUN-2). The hippocratic screening and biochemical analysis were performed in Balb/c male mice that received a high dose of each compound, and the compounds showed no in vivo toxicity. The treatment of experimental PCM with the new oxadiazoles led to significant reduction in CFU (≥1 log10). Histopathological analysis of the groups treated exhibited control of inflammation, as well as preserved lung areas. These findings suggest that LMM5 and LMM11 are promising hits structures, opening the door for implementing new PCM therapies.

Promising New Antifungal Treatment Targeting Chorismate Synthase from Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM), caused by Paracoccidioides, is a systemic mycosis with granulomatous character and a restricted therapeutic arsenal. The aim of this work was to search for new alternatives to treat largely neglected tropical mycosis, such as PCM. In this context, the enzymes of the shikimate pathway constitute excellent drug targets for conferring selective toxicity because this pathway is absent in humans but essential for the fungus. In this work, we have used a homology model of the chorismate synthase (EC 4.2.3.5) from Paracoccidioides brasiliensis (PbCS) and performed a combination of virtual screening and molecular dynamics testing to identify new potential inhibitors. The best hit, CP1, successfully adhered to pharmacological criteria (adsorption, distribution, metabolism, excretion, and toxicity) and was therefore used in in vitro experiments. Here we demonstrate that CP1 binds with a dissociation constant of 64 ± 1 µM to recombinant chorismate synthase from P. brasiliensis and inhibits enzymatic activity, with a 50% inhibitory concentration (IC50) of 47 ± 5 µM. As expected, CP1 showed no toxicity in three cell lines. On the other hand, CP1 reduced the fungal burden in lungs from treated mice, similar to itraconazole. In addition, histopathological analysis showed that animals treated with CP1 displayed less lung tissue infiltration, fewer yeast cells, and large areas with preserved architecture. Therefore, CP1 was able to control PCM in mice with a lower inflammatory response and is thus a promising candidate and lead structure for the development of drugs useful in PCM treatment.

Propolis for the Treatment of Onychomycosis.

Onychomycosis is a fungal nail infection, considered as a public health problem because it is contagious and it interferes with the quality of life. It has long and difficult treatment, with many side effects and high cost. Propolis extract (PE) is a potential alternative to conventional antifungal agents because it has low cost, accessibility, and low toxicity. Herein, we report the favorable response of PE in onychomycosis in three elderly patients.

In Vitro Control of Uropathogenic Microorganisms with the Ethanolic Extract from the Leaves of Cochlospermum regium (Schrank) Pilger.

The roots of Cochlospermum regium, popularly known as "algodãozinho-do-cerrado," are used for the treatment of genitourinary infections. However, the removal of their subterranean structures results in the death of the plant, and the use of the leaves becomes a viable alternative. Therefore, the antimicrobial activity of Cochlospermum regium leaf's ethanolic extract and its action on the biofilm formation of microorganisms associated with urinary infection were evaluated. The total phenolic compounds, flavoids, and tannins were quantified using the reagents Folin-Ciocalteu, aluminum chloride, and vanillin, respectively. The antimicrobial activity was evaluated by the broth microdilution method and the effect of the extract in the biofilm treatment was measured by the drop plate method. Cytotoxicity was evaluated by the method based on the reduction of MTS and the mutagenicity by the Ames test. The ethanolic extract of C. regium leaves presented 87.4 mg/EQ of flavonoids, 167.2 mg/EAG of total phenolic compounds, and 21.7 mg/ECA of condensed tannins. It presented reduction of the biofilm formation for E. coli and C. tropicalis and antimicrobial action of 1 mg/mL and 0.5 mg/mL, respectively. The extract showed no cytotoxicity and mutagenicity at the concentrations tested. This study demonstrated that C. regium leaves are a viable option for the treatment of genitourinary infections and for the species preservation.

Candida albicans PROTEIN PROFILE CHANGES IN RESPONSE TO THE BUTANOLIC EXTRACT OF Sapindus saponariaL.

Candida albicans is an opportunistic human pathogen that is capable of causing superficial and systemic infections in immunocompromised patients. Extracts of Sapindus saponaria have been used as antimicrobial agents against various organisms. In the present study, we used a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify the changes in protein abundance of C. albicans after exposure to the minimal inhibitory concentration (MIC) and sub-minimal inhibitory concentration (sub-MIC) of the butanolic extract (BUTE) of S. saponaria and also to fluconazole. A total of six different proteins with greater than 1.5 fold induction or repression relative to the untreated control cells were identified among the three treatments. In general, proteins/enzymes involved with the glycolysis (GPM1, ENO1, FBA1), amino acid metabolism (ILV5, PDC11) and protein synthesis (ASC1) pathways were detected. In conclusion, our findings reveal antifungal-induced changes in protein abundance of C. albicans. By using the previously identified components of the BUTE of S. saponaria(e.g., saponins and sesquiterpene oligoglycosides), it will be possible to compare the behavior of compounds with unknown mechanisms of action, and this knowledge will help to focus the subsequent biochemical work aimed at defining the effects of these compounds.

ANTIFUNGAL POTENTIAL OF PLANT SPECIES FROM BRAZILIAN CAATINGA AGAINST DERMATOPHYTES.

Trichophyton rubrum and Trichophyton mentagrophytes complex, or Trichophyton spp. are the main etiologic agents of dermatophytosis, whose treatment is limited by the high cost of antifungal treatments, their various side effects, and the emergence of resistance amongst these species. This study evaluated the in vitro antidermatophytic activity of 23 crude extracts from nine plant species of semiarid vegetation (caatinga) found in Brazil. The extracts were tested at concentrations ranging from 1.95 to 1,000.0 mg/mL by broth microdilution assay against the reference strains T. rubrum ATCC 28189 and T. mentagrophytes ATCC 11481, and 33 clinical isolates of dermatophytes. All plants showed a fungicidal effect against both fungal species, with MIC/MFC values of the active extracts ranging from 15.6 to 250.0 µg/mL. Selected extracts of Eugenia uniflora (AcE), Libidibia ferrea (AE), and Persea americana (AcE) also exhibited a fungicidal effect against all clinical isolates of T. rubrum and T. mentagrophytes complex. This is the first report of the antifungal activity of Schinus terebinthifolius, Piptadenia colubrina, Parapiptadenia rigida, Mimosa ophthalmocentra, and Persea americana against both dermatophyte species.

Effect of highly active antiretroviral therapy on vaginal Candida spp. isolation in HIV-infected compared to HIV-uninfected women.

Vulvovaginal candidiasis (VVC) in HIV-infected women contributed to the impairment of their quality of life. The aim of this study was to evaluate the effect of highly active antiretroviral therapy (HAART) use on the vaginal Candida spp. isolation in HIV-infected compared to HIV-uninfected women. This cross-sectional study included 178 HIV-infected (HIV group) and 200 HIV-uninfected women (control) that were studied at the Specialized Assistance Service (SAE) for sexually transmitted diseases (STD)/AIDS of the city of Maringá, Brazil, from April 1 to October 30, 2011. The yeasts were isolated and identified by phenotypic and molecular methods. The in vitro antifungal susceptibility to fluconazole, itraconazole, nystatin and amphotericin B was tested by the reference microdilution method. Higher frequencies of total vaginal Candida spp. isolation were found in the HIV-infected group than in the control group. However, both groups showed a similar frequency of colonization and VVC. Although C. albicans was the most frequent and sensitive to azolics and polyenes in both HIV-infected and uninfected women, the emerging resistance of C. glabrata to amphotericin B in the HIV-infected women was observed. Although higher frequency of vaginal Candida spp. isolation had been observed in the HIV-infected than in HIV-uninfected women, colonization and VVC showed similar frequency in both groups, indicating that HAART appears to protect against vaginal colonization and VVC.

Propolis Is an Efficient Fungicide and Inhibitor of Biofilm Production by Vaginal Candida albicans.

Vulvovaginal candidiasis (VVC) is one of the most common genital infections in women. The therapeutic arsenal remains restricted, and some alternatives to VVC treatment are being studied. The present study evaluated the influence of a propolis extractive solution (PES) on biofilm production by Candida albicans isolated from patients with VVC. Susceptibility testing was used to verify the minimum inhibitory concentration (MIC) of PES, with fluconazole and nystatin as controls. The biofilm formation of 29 vaginal isolates of C. albicans and a reference strain that were exposed to PES was evaluated using crystal violet staining. Colony-forming units were evaluated, proteins and carbohydrates of the matrix biofilm were quantified, and scanning electron microscopy was performed. The MIC of PES ranged from 68.35 to 546.87 µg/mL of total phenol content in gallic acid. A concentration of 546.87 µg/mL was able to cause the death of 75.8% of the isolates. PES inhibited biofilm formation by C. albicans from VVC. Besides antifungal activity, PES appears to present important antibiofilm activity on abiotic surfaces, indicating that it may have an additional beneficial effect in the treatment of VVC.

ANTIFUNGAL ACTIVITY OF Cymbopogon nardus (L.) Rendle (CITRONELLA) AGAINST Microsporum canis FROM ANIMALS AND HOME ENVIRONMENT.

Dermatophytosis is a common zoonosis in urban centers. Dogs and cats have played an important role as its disseminators. Environmental decontamination is essential for the prevention of its propagation to humans and animals. However, sanitizers or disinfectants with antifungal activity, currently available, have high toxicity. The present study evaluated the in vitro effects of an extract of citronella (Cymbopogon nardus) on 31 Microsporum canis isolates from animals and home environments. Susceptibility tests were performed based on document M38-A2 (2008) of the Clinical and Laboratory Standards Institute with modifications for natural products. Although susceptibility variation was observed between the fungus tested, the concentrations that inhibited the growth of 50 and 90% of the microorganisms were low (19.5 and 78 µg/mL, respectively). Thus, this citronella extract showed potent fungistatic and fungicide activities against M. canis isolated from animals and home environments. Therefore, it could be an alternative for dermatophytosis prophylaxis in the home environment.

In vitro effect of Paullinia cupana (guaraná) on hydrophobicity, biofilm formation, and adhesion of Candida albicans' to polystyrene, composites, and buccal epithelial cells.

OBJECTIVE: In vitro evaluation of the effect of guaraná (GUAR) on cell surface hydrophobicity (CSH), on biofilm formation, and on adhesion of C. albicans to polystyrene, to composite resins, and to buccal epithelial cells (BEC). MATERIALS AND METHODS: Lyophilised aqueous extract of GUAR was tested on C. albicans ATCC (90028). The effect of GUAR was evaluated by examining the CSH of C. albicans, as determined by microbial adhesion to hydrocarbons test, by assessing biofilm production and through adhesion assays (microplates of polystyrene, BEC and composites). One nanoparticle (Z350(®)) and two microhybrid (LLis(®), Opallis(®)) composites were tested. Scanning electron microscopy (SEM) was used to analyse adhesion of C. albicans composites. Assays were performed in triplicate and the results analysed by Chi-square test, Kruskal-Wallis test and Dunn's Multiple Comparison post hoc test at 5% significance level. RESULTS: GUAR did not inhibit growth of C. albicans at any concentration, but it reduced adhesion to polystyrene surface (p < 0.001). Exposure to GUAR did not change CSH and biofilm formation, but it increased adhesion of C. albicans to the nanoparticle composite (p = 0.042) and reduced its adhesion to BEC (p < 0.001). SEM confirmed an aggregatory pattern of adhesion of C. albicans to composites. CONCLUSION: GUAR increased the adhesion of C. albicans to the surface of the nanoparticle composite. However, it reduced the adhesion of C. albicans to BEC and to polystyrene, which reveals its potential use in prevention of oral diseases.

Antifungal Activity of Brazilian Propolis Microparticles against Yeasts Isolated from Vulvovaginal Candidiasis.

Propolis, a resinous compound produced by Apis mellifera L. bees, is known to possess a variety of biological activities and is applied in the therapy of various infectious diseases. The aim of this study was to evaluate the in vitro antifungal activity of propolis ethanol extract (PE) and propolis microparticles (PMs) obtained from a sample of Brazilian propolis against clinical yeast isolates of importance in the vulvovaginal candidiasis (VVC). PE was used to prepare the microparticles. Yeast isolates (n = 89), obtained from vaginal exudates of patients with VVC, were exposed to the PE and the PMs. Moreover, the main antifungal drugs used in the treatment of VVC (Fluconazole, Voriconazole, Itraconazole, Ketoconazole, Miconazole and Amphotericin B) were also tested. Minimum inhibitory concentration (MIC) was determined according to the standard broth microdilution method. Some Candida albicans isolates showed resistance or dose-dependent susceptibility for the azolic drugs and Amphotericin B. Non-C. albicans isolates showed more resistance and dose-dependent susceptibility for the azolic drugs than C. albicans. However, all of them were sensitive or dose-dependent susceptible for Amphotericin B. All yeasts were inhibited by PE and PMs, with small variation, independent of the species of yeast. The overall results provided important information for the potential application of PMs in the therapy of VVC and the possible prevention of the occurrence of new symptomatic episodes.