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Aim: Glycosphingolipids are conserved lipids displaying a variety of functions in fungal cells, such as determination of cell polarity and virulence. They have been considered as potent targets for new antifungal drugs. The present work aimed to test two inhibitors, myriocin and DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol, in Scedosporium boydii , a pathogenic fungus which causes a wide range of disease. Materials & methods: Mass spectrometry, microscopy and cell biology approaches showed that treatment with both inhibitors led to defects in fungal growth and membrane integrity, and caused an increased susceptibility to the current antifungal agents. Conclusion: These data demonstrate the antifungal potential of drugs inhibiting sphingolipid biosynthesis, as well as the usefulness of sphingolipids as promising targets for the development of new therapeutic options.

Biofilms are frequently related to invasive fungal infections and are reported to be more resistant to antifungal drugs than planktonic cells. The structural complexity of the biofilm as well as the presence of a polymeric extracellular matrix (ECM) is thought to be associated with this resistant behavior. Scanning electron microscopy (SEM) after room temperature glutaraldehyde-based fixation, have been used to study fungal biofilm structure and drug susceptibility but they usually fail to preserve the ECM and, therefore, are not an optimised methodology to understand the complexity of the fungal biofilm. Thus, in this work, we propose a comparative analysis of room-temperature and cryofixation/freeze substitution of Candida albicans biofilms for SEM observation. Our experiments showed that room-temperature fixative protocols using glutaraldehyde and osmium tetroxide prior to alcohol dehydration led to a complete extraction of the polymeric ECM of biofilms. ECM from fixative and alcohol solutions were recovered after all processing steps and these structures were characterised by biochemistry assays, transmission electron microscopy and mass spectrometry. Cryofixation techniques followed by freeze-substitution lead to a great preservation of both ECM structure and C. albicans biofilm cells, allowing the visualisation of a more reliable biofilm structure. These findings reinforce that cryofixation should be the indicated method for SEM sample preparation to study fungal biofilms as it allows the visualisation of the EMC and the exploration of the biofilm structure to its fullest, as its structural/functional role in interaction with host cells, other pathogens and for drug resistance assays., (© 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.)

Candida spp. can adhere to and form biofilms over different surfaces, becoming less susceptible to antifungal treatment. Resistance of biofilms to antifungal agents is multifactorial and the extracellular matrix (ECM) appears to play an important role. Among the few available antifungals for treatment of candidaemia, only the lipid formulations of amphotericin B (AmB) and the echinocandins are effective against biofilms. Our group has previously demonstrated that miltefosine has an important effect against Candida albicans biofilms. Thus, the aim of this work was to expand the analyses of the in vitro antibiofilm activity of miltefosine to non-albicans Candida spp. Miltefosine had significant antifungal activity against planktonic cells and the development of biofilms of C. albicans, Candida parapsilosis, Candida tropicalis and Candida glabrata. The activity profile in biofilms was superior to fluconazole and was similar to that of AmB and caspofungin. Biofilm-derived cells with their ECM extracted became as susceptible to miltefosine as planktonic cells, confirming the importance of the ECM in the biofilm resistant behaviour. Miltefosine also inhibited biofilm dispersion of cells at the same concentration needed to inhibit planktonic cell growth. The data obtained in this work reinforce the potent inhibitory activity of miltefosine on biofilms of the four most pathogenic Candida spp. and encourage further studies for the utilisation of this drug and/or structural analogues on biofilm-related infections., (Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

The generation of a new antifungal against Candida albicans biofilms has become a major priority, since biofilm formation by this opportunistic pathogenic fungus is usually associated with an increased resistance to azole antifungal drugs and treatment failures. Miltefosine is an alkyl phospholipid with promising antifungal activity. Here, we report that, when tested under planktonic conditions, miltefosine displays potent in vitro activity against multiple fluconazole-susceptible and -resistant C. albicans clinical isolates, including isolates overexpressing efflux pumps and/or with well-characterized Erg11 mutations. Moreover, miltefosine inhibits C. albicans biofilm formation and displays activity against preformed biofilms. Serial passage experiments confirmed that miltefosine has a reduced potential to elicit resistance, and screening of a library of C. albicans transcription factor mutants provided additional insight into the activity of miltefosine against C. albicans growing under planktonic and biofilm conditions. Finally, we demonstrate the in vivo efficacy of topical treatment with miltefosine in the murine model of oropharyngeal candidiasis. Overall, our results confirm the potential of miltefosine as a promising antifungal drug candidate, in particular for the treatment of azole-resistant and biofilm-associated superficial candidiasis., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Onychomycosis is a fungal nail infection that represents ∼50 % of all nail disease cases worldwide. Clinical treatment with standard antifungals frequently requires long-term systemic therapy to avoid chronic disease. Onychomycosis caused by non-dermatophyte moulds, such as Fusarium spp., and yeasts, such as Candida spp., is particularly difficult to treat, possibly due to the formation of drug-resistant fungal biofilms on affected areas. Here, we show that the alkylphospholipid miltefosine, used clinically against leishmaniasis and cutaneous breast metastases, has potent activity against biofilms of Fusarium oxysporum and Candida albicans formed on human nail fragments in vitro. Miltefosine activity was compared with that of commercially available antifungals in the treatment of biofilms at two distinct developmental phases: formation and maturation (pre-formed biofilms). Drug activity towards biofilms formed on nail fragments and on microplate surfaces (microdilution assays) was evaluated using XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assays, and drug effects on fingernail biofilms were analysed by scanning electron microscopy (SEM). For F. oxysporum, miltefosine at 8 μg ml- 1 inhibited biofilm formation by 93%, whilst 256 μg ml- 1 reduced the metabolic activity of pre-formed nail biofilms by 93%. Treatment with miltefosine at 1000 μg ml- 1 inhibited biofilm formation by 89% and reduced the metabolic activity of pre-formed C. albicans biofilms by 99%. SEM analyses of biofilms formed on fingernail fragments showed a clear reduction in biofilm biomass after miltefosine treatment, in agreement with XTT results. Our results show that miltefosine has potential as a therapeutic agent against onychomycosis and should be considered for in vivo efficacy studies, especially in topical formulations for refractory disease treatment.

Onychomycoses represent approximately 50 % of all nail diseases worldwide. In warmer and more humid countries like Brazil, the incidence of onychomycoses caused by non-dermatophyte molds (NDM, including Fusarium spp.) or yeasts (including Candida albicans) has been increasing. Traditional antifungal treatments used for the dermatophyte-borne disease are less effective against onychomycoses caused by NDM. Although some laser and light treatments have demonstrated clinical efficacy against onychomycosis, their US Food and Drug Administration (FDA) approval as "first-line" therapy is pending, partly due to the lack of well-demonstrated fungicidal activity in a reliable in vitro model. Here, we describe a reliable new in vitro model to determine the fungicidal activity of laser and light therapies against onychomycosis caused by Fusarium oxysporum and C. albicans. Biofilms formed in vitro on sterile human nail fragments were treated with 1064 nm neodymium-doped yttrium aluminum garnet laser (Nd:YAG), 420 nm intense pulsed light (IPL) IPL 420, followed by Nd:YAG, or near-infrared light ((NIR) 700-1400 nm). Light and laser antibiofilm effects were evaluated using cell viability assay and scanning electron microscopy (SEM). All treatments were highly effective against C. albicans and F. oxysporum biofilms, resulting in decreases in cell viability of 45-60 % for C. albicans and 92-100 % for F. oxysporum. The model described here yielded fungicidal activities that matched more closely to those observed in the clinic, when compared to published in vitro models for laser and light therapies. Thus, our model might represent an important tool for the initial testing, validation, and "fine-tuning" of laser and light therapies against onychomycosis.

Background: Biofilm formation is important in Candida albicans pathogenesis and constitutes a mechanism of antifungal resistance. Thus, we evaluated the effect of proanthocyanidins polymer-rich fractions from Stryphnodendron adstringens (fraction F2 and subfraction F2.4) against C. albicans biofilms., Methods: Firstly, the antifungal activity of F2 and F2.4 against planktonic cells of Candida albicans (ATCC 10231) was determined using broth microdilution method. Anti-biofilm effect of F2 and F2.4 was evaluated during biofilm formation or on mature biofilm of C. albicans and compared with standard antifungals amphotericin B and fluconazole. Metabolic activity of sessile and dispersion cells from biofilms after antifungal treatments were measured using a tetrazolium reduction assay and the biofilm total biomass was quantified by crystal violet-based assay. Morphological alterations after treatments were observed using scanning electron microscopy., Results: The anti-biofilm effect of F2 and F2.4 were comparable to standard antifungals (amphotericin B and fluconazole). F2 and F2.4 treatments reduced biofilm metabolic activity (in sessile and in dispersion cells) during biofilm formation, and in mature biofilms, unlike fluconazole, which only prevents the biofilm formation. Treatments with F2, F2.4 or fluconazole reduced biofilm biomass during biofilm formation, but not in mature biofilm. Amphotericin B presented higher inhibitory effect on biofilm formation and on mature biofilm of C. albicans. F2 and F2.4 treatments led to the appearance of dumbbell-shaped blastoconidia and of blastoconidia clusters in biofilms., Conclusion: Proanthocyanidins polymer-rich fractions from S. adstringens successfully inhibited C. albicans planktonic growth and biofilm development, and they represent a potential new agent for the treatment of biofilm-associated candidiasis.

Objectives: The aim of this study was to evaluate miltefosine and four synthetic compounds (TCAN26, TC19, TC106 and TC117) for their in vitro inhibitory activity against Candida albicans planktonic and biofilm cells and investigate whether these compounds are able to inhibit the biofilm formation and to reduce the viability of mature C. albicans biofilm cells., Methods: The XTT reduction assay and transmission and scanning electron microscopy were employed to determine the inhibitory effects of the test compounds in comparison with amphotericin B and fluconazole against both planktonic cells and sessile cells in biofilms., Results: C. albicans planktonic cells were susceptible to miltefosine, TCAN26 and TC19, all alkylphospholipid compounds. Miltefosine and TCAN26 present a fungicidal activity with similar values of MIC and minimum fungicidal concentration (MFC), ranging from 2 to 8 mg/L. Cell treatment with sub-inhibitory concentrations of alkylphospholipids induced several ultrastructural alterations. In relation to biofilms, miltefosine reduced formation (38%-71%) and mature biofilms viability (32%-44%), at concentrations of 64 mg/L. TCAN26 also reduced biofilm formation (24%-30%) and mature biofilm viability (15%-20%), at concentrations of 64 mg/L. Although amphotericin B reduced biofilm formation similarly to miltefosine (51%-74%), its activity was lower on mature biofilms (24%-30%). Miltefosine antibiofilm activity was significantly higher than amphotericin B, on both formation and mature biofilms (P<0.05 and P<0.0001, respectively). Fluconazole was the least effective compound tested., Conclusion: Promising antibiofilm activity was displayed by miltefosine and other alkylphosphocholine compounds, which could be considered a putative option for future treatment of candidaemia associated with biofilm formation, although further evaluation in in vivo systems is required.