Detection of 2-ethyl-1-hexanol and its modulating effect in biofilm of Fusarium oxysporum.

In immunocompetent individuals, Fusarium spp. stands out as the causative agent of onychomycosis, among the non-dermatophyte molds. Despite evidence indicating that Fusarium oxysporum organizes itself in the form of a biofilm causing onychomycosis, there is little literature on the etiopathogenesis of the biofilm on the nail, specifically the signaling molecules present, known as quorum sensing (QS). Thus, this study detected the presence of a molecule related to QS from the ex vivo biofilm of F. oxysporum on human nail and investigated its effect on preformed biofilm in vitro. The detection and physicochemical characterization of a QS molecule, from the extracellular matrix (ECM), was carried out by Fourier transform infrared (FTIR) spectroscopy with an attenuated total reflectance (ATR) accessory and by headspace gas chromatography coupled to mass spectrometry (GC-MS) analyses. Determination of viable cells, cell activity, total biomass, ECM components and scanning electron microscopy (SEM) were performed to evaluate the influence of the QS molecule on the in vitro biofilm of F. oxysporum. The beginning, in the ex vivo biofilm of F. oxysporum on human nails, the volatile organic compound 2-ethyl-1-hexanol (2EH) was detected as a component of QS. Thereafter in vitro analyses, synthetic 2EH was able to modulate the biofilm by stimulating its filament, increasing total biomass and ECM production in terms of total carbohydrates, but with a reduction in total proteins and nucleic acids. We thus evidence, for the first time, the presence of 2EH in the biofilm of F. oxysporum, developed on the human nail, and the in vitro action of this compound as a QS molecule.

Photoactivity of hypericin: from natural product to antifungal application.

Considering the multifaceted and increasing application of photodynamic therapy (PDT), in recent years the antimicrobial employment of this therapy has been highlighted, because of the antiviral, antibacterial, antiparasitic, and antifungal activities that have already been demonstrated. In this context, research focussed on antimycological action, especially for treatment of superficial infections, presents promising growth due to the characteristics of these infections that facilitate PDT application as new therapeutic options are needed in the field of medical mycology. Among the more than one hundred classes of photosensitizers the antifungal action of hypericin (Hyp) stands out due to its ability to permeate the lipid membrane and accumulate in different cytoplasmic organelles of eukaryotic cells. In this review, we aim to provide a complete overview of the origin, physicochemical characteristics, and optimal alternative drug deliveries that promote the photodynamic action of Hyp (Hyp-PDT) against fungi. Furthermore, considering the lack of a methodological consensus, we intend to compile the best strategies to guide researchers in the antifungal application of Hyp-PDT. Overall, this review provides a future perspective of new studies and clinical possibilities for the advances of such a technique in the treatment of mycoses in humans.

The impact of ORF19.36.1 in the pathobiology of Candida albicans.

BACKGROUND: Our previous proteomics data obtained from Candida albicans recovered after serial passage in a murine model of systemic infection revealed that Orf19.36.1 expression correlates with the virulence of the fungus. Therefore, the impact of ORF19.36.1 upon virulence was tested in this study. MATERIALS & METHODS: CRISPR-Cas9 technology was used to construct homozygous C. albicans orf19.36.1 null mutants and the phenotypes of these mutants examined in vitro (filamentation, invasion, adhesion, biofilm formation, hydrolase activities) and in vivo assays. RESULTS: The deletion of ORF19.36.1 did not significantly impact the phenotypes examined or the virulence of C. albicans in two infection models. CONCLUSION: These results suggest that, although Orf19.36.1 expression correlates with virulence, this protein is not essential for C. albicans pathobiology.

In vitro ability of Fusarium keratoplasticum to form biofilms in venous catheter.

Fusarium keratoplasticum is a common specie in human infections and is responsible for many diseases affecting immunocompetent and immunocompromised patients. This study aimed to evaluate the ability of Fusarium keratoplasticum to form biofilm in venous catheters (VC), focusing on the development of maturation and dispersion over time (24, 48, 72, 96 and 120 h) and the evaluation amphotericin B (AB) susceptibility in planktonic cells and after 96 h of biofilm formation. F. keratoplasticum was able to form a biofilm in VC with maturation most likely between 48 and 72 h, according to colony count and total biomass results. The dispersion process supposedly occurred from 72 to 96 h, when we observed a decrease in the parameter's colony count, total biomass and mitochondrial metabolic activity. The planktonic cells of F. keratoplasticum were susceptible to AB, however, there was no inhibition of the F. keratoplasticum strain biofilm in any of the AB concentrations, with the growth of the fungus recovering after 48 h in contact with AB. Thus, our findings suggest that in addition to forming a biofilm on VC, F. keratoplasticum becomes AB-resistant, highlighting the concern of this fungus on medical devices.

Relevant insights into onychomycosis' pathogenesis related to the effectiveness topical treatment.

Onychomycosis (OM) is a fungal infection, responsible for about 50% of nail diseases. OM has been attributed to the ability of fungi to naturally organize themselves into biofilms on nail surfaces. However, little is known about the exact role of the biofilm in the etiopathogenesis of OM, as well as its influence in the permeation of a topical treatment. The objectives of this study were to review the literature for topical OM treatments in clinical trials, assess the efficiency of these treatments, and discuss factors that could affect the success of these treatments. First, a systematic search of articles published in the MEDLINE database (PubMed) between January 2010 and December 2019 was conducted, focusing on drugs under clinical trials for the topical treatment of OM. Of the publications selected, it was clear that none of them had considered the fungi organized in biofilm. Therefore, we reflected on some important variables involved in OM, such as the nail structure and the mechanism of fungal invasion. Some methods, such as histopathologic analysis and spectroscopy techniques, were found to be effective in the detection of nail biofilm, and could be used in future drug permeation studies. This review allowed us to conclude that novel antifungals for the topical treatment of OM must consider the drug to permeate through biofilm. Natural products, such as propolis, seem strong candidates in this respect.

Insights on the etiopathogenesis of onychomycosis by dermatophyte, yeast and non-dermatophyte mould in ex vivo model.

Fungal biofilms have been involved in the pathogenesis of onychomycosis, but the aspects contributing to this association need to be enlightened. This study aimed to investigate the ability of three different fungi to form biofilm on the nail. All evaluated fungi were able to grow on the nails, using them as the only nutritional source and formed a structure strongly suggestive of biofilms. However, their architecture and morphology were highly contrasting: Candida albicans showed dense growth, exhibited a well-structured community and a large amount of extracellular matrix (ECM), and FTIR-ATR spectroscopy reinforced these findings revealing components suggestive of the biofilm. For Fusarium oxysporum, these events were also observed, but in lower intensity. Furthermore, while Trichophyton rubrum presented a well-organized architecture, the ECM was not visualized. We hypothesize that these findings are related to the symptomatology of onychomycosis. When it is caused by a non-dermatophyte, it usually is accompanied with paronychia, pain, oedema, inflammation and few signals of keratolysis, while dermatophytes are more associated with intense onycholysis and absence of the inflammatory signals. Biofilm seems to be crucial for non-dermatophytes to cause onychomycosis, but not for T. rubrum.

Rhodotorula sp. and Trichosporon sp. are more Virulent After a Mixed Biofilm.

Rhodotorula spp. and Trichosporon spp. are opportunistic pathogens, and although an association between these two species in the same infection appears to be uncommon, it has been reported. This is the first study that aimed to evaluate the pathogenesis of a co-infection by R. mucilaginosa and T. asahii, using a new in vivo model, the Zophobas morio larvae. Suspensions from planktonic and biofilm-recovered cells were injected in the larvae as in monospecies as mixed (a ratio of 1:1 for both agents of a of 105 inoculum). Individual and mixed biofilms of R. mucilaginosa and T. asahii were produced for 24 and 48 h, and they were partially characterized by crystal violet and reduction of tetrazolium salt. When evaluating the impact of the planktonic suspension in vivo we verified that the fungi in monoculture were more able to kill the larvae than those from planktonic mixed suspension. On the other hand, regarding biofilm-recovered cells, there was an increase in the death of larvae infected for mixed suspensions. Moreover, the death rate was more pronounced when the larvae were infected with 48 h biofilm-recovered cells than the 24 h ones. T. asahii was the best producer of total biomass, mainly in 48 h. The metabolic activity for both yeasts organized in biofilm maintained the same pattern between 24 and 48 h. The present study proves a synergistic interaction between R. mucilaginosa and T. asahii after an experience in a mixed biofilm. Our results suggest that both species were benefited from this interaction, acquiring a greater potential for virulence after passing through the biofilm and this ability was acquired by the cells released from the biofilm.

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.

The ability of farnesol to prevent adhesion and disrupt Fusarium keratoplasticum biofilm.

A biofilm is represented by a community of microorganisms capable of adhering to a surface and producing substances that envelop the cells, forming an extracellular matrix. The extracellular matrix is responsible for protecting microorganisms against environmental stress, hosts the immune system and confers resistance to antimicrobials. Fusarium keratoplasticum is a common species of FSSC (Fusarium solani species complex) associated with human infections, being the most prevalent species related to biofilm formation in hospital water systems and internal pipelines. With this in mind, this study aimed to characterise the biofilm formed by the fungus F. keratoplasticum and to evaluate the effects of farnesol, a fungal quorum sensing (QS) molecule, on the preformed biofilm and also during its formation at different times (adhesion and 24, 48 and 72 h). F. keratoplasticum is able to adhere to an abiotic surface and form a dense biofilm in 72 h, with increased total biomass and matrix modulation with the presence of extracellular DNA, RNA, polysaccharides and proteins. Farnesol exhibited important anti-biofilm activity, causing the destruction of hyphae and the extracellular matrix in preformed biofilm and preventing the adhesion of conidia, filamentation and the formation of biofilm. Few studies have characterised the formation of biofilm by filamentous fungi. Our findings suggest that farnesol acts efficiently on F. keratoplasticum biofilm since this molecule is capable of breaking the extracellular matrix, thereby disarranging the biofilm.

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.

Effects of intratracheal Fusarium solani inoculation in immunocompetent mice.

Fusariosis is an infection that is caused by fungi of the Fusarium genus. It is the second most common fungus that is associated with human fungal infections, usually in immunocompromised individuals. The incidence of such infections has been increasing, including in immunocompetent hosts. Studies of host-pathogen interactions are scarce, and the pathophysiology of the disease is unknown. One limitation of such studies is the lack of adequate techniques for mammalian infection, in which no standardized protocols have been established with fungi with a focus on the respiratory tract. The aim of the present study was to assess the first 24 h of infection after the intratracheal inoculation of F. solani microconidia in immunocompetent mice. Colony-forming units (CFU) were counted, and histopathological analysis was performed. Under conditions of high fungal burden, F. solani caused lethal tissue damage in the lungs. Under conditions of low fungal burden, the infection was not lethal, but several alterations of pulmonary tissue and the presence of the fungus in the lungs were observed. No evidence of fungal dissemination was found in the kidneys, spleen, liver, or heart 24 h after infection. The present intratracheal model effectively established fungal infection and appears to be suitable for studies of Fusarium spp.

Repurposing approach identifies new treatment options for invasive fungal disease.

Drug repositioning is the process of discovery, validation and marketing of previously approved drugs for new indications. Our aim was drug repositioning, using ligand-based and structure-based computational methods, of compounds that are similar to two hit compounds previously selected by our group that show promising antifungal activity. Through the ligand-based method, 100 compounds from each of three databases (MDDR, DrugBank and TargetMol) were selected by the Tanimoto coefficient, as similar to LMM5 or LMM11. These compounds were analyzed by the scaffold trees, and up to 10 compounds from each database were selected. The structure-based method (molecular docking) using thioredoxin reductase as the target drug was performed as a complementary approach, resulting in six compounds that were tested in an in vitro assay. All compounds, particularly raltegravir, showed antifungal activity against the genus Paracoccidioides. Raltegravir, an antiviral drug, showed promising antifungal activity against the experimental murine paracoccidioidomycosis, with significant reduction of the fungal burden and decreased alterations in the lung structure of mice treated with 1 mg/kg of raltegravir. In conclusion, the combination of two in silico methods for drug repositioning was able to select an antiviral drug with promising antifungal activity for treatment of paracoccidioidomycosis.

Selection of potential anti-adhesion drugs by in silico approaches targeted to ALS3 from Candida albicans.

OBJECTIVE: To select potential ligands of ALS3 for drug development with anti-adhesion and/or anti-biofilm activities. METHODOLOGY: ALS3 model was considered stable by DM. The main features of protein flexibility were represented by two conformers which were used in the virtual screening. Twenty-four small molecules were selected for in vitro assays. Five of them presented the best biological activity with ability to inhibit the adhesion and C. albicans biofilm formation on abiotic surface. RESULTS: To select potential ligands of ALS3 for drug development with anti-adhesion and/or anti-biofilm activities. CONCLUSION: In silico tools application was able to select promising compounds with anti-adhesion activity, opening a new perspective of medical device treatment.

Yeasts from skin colonization are able to cross the acellular dermal matrix.

In recent decades, the prognosis for burn patients has improved considerably with the development of specialized care. The acellular dermal matrix (ADM) is a totally artificial acellular device that functions to control water loss, prevent penetration by bacteria and allow migration of endothelial cells and fibroblasts from patient tissues. However, little is known about its effectiveness against yeasts. The present study evaluated the capacity of colonization and migration of some human commensal yeasts. Three clinical isolates from skin scales, identified as Candida parapsilosis, Candida glabrata and Rhodotorula mucilaginosa, were used. Their ability to cross the ADM was evaluated. After three days, all isolates had crossed the ADM. C. parapsilosis showed the lowest growth, while R. mucilaginosa showed intermediate and C. glabrata the highest growth. In the plates incubated for seven days, the growth of C. parapsilosis and C. glabrata increased by 1 log over the third day. All isolates have the capacity to colonize and migrate through the matrix, increasing the potential risk to burn patients, who can develop severe and even fatal infections by invasive fungi.

Murine model for the evaluation of candiduria caused by Candida tropicalis from biofilm.

To evaluate the pathophysiology of catheter-associated candiduria, the bladders of female mice were infected with Candida tropicalis. One group was implanted with a catheter fragment with preformed biofilm by cystotomy technique, while another group received, in separate, a sterile catheter fragment and a correspondent yeast suspension. The bladder tissues were examined by histopathology and the quantity of colony forming units was evaluated. All the animals presented inflammation and the presence of C. tropicalis was observed in the tissue within 72 h of the introduction of biofilm, while 75% of the mice remained infected after 144 h. However, only 50% of animals from the group infected with C. tropicalis in suspension (planktonic yeasts), exhibited such signs of infection over time. The cystotomy technique is therefore viable in mice, and is an effective model for evaluating the pathogenesis of candiduria from catheter biofilms. The model revealed the potential of C. tropicalis infectivity and demonstrated more effective evasion of the host response in biofilm form than the planktonic yeast.

Thermal Magnetic Field Activated Propolis Release From Liquid Crystalline System Based on Magnetic Nanoparticles.

Intra-periodontal pocket drug delivery systems, such as liquid crystalline systems, are widely utilized improving the drug release control and the therapy. Propolis is used in the treatment of periodontal diseases, reducing the inflammatory and infectious conditions. Iron oxide magnetic nanoparticles (MNPs) can improve the treatment when an alternating external magnetic field (AEMF) is applied, increasing the local temperature. The aim of this study was to develop a liquid crystalline system containing MNPs for intra-periodontal pocket propolis release. MNPs were prepared using iron salts and the morphological, size, thermal, x-ray diffraction, magnetometry, and Mössbauer spectroscopy analyses were performed. Cytotoxicity studies using Artemia salina and fibroblasts were also accomplished. The systems were prepared using polyoxyethylene (10) oleyl ether, isopropyl myristate, purified water, and characterized by polarized optical microscopy, rheometry, and in vitro drug release profile using a periodontal pocket simulator apparatus. The antifungal activity of the systems was investigated against Candida spp. using an AEMF. MNPs displayed nanometric size, were monodisperse, and they displayed very low cytotoxicity. Microscopically homogeneous formulations were obtained displaying important physicochemical and biological properties. The system displayed prolonged release of propolis and important in vitro fungicide activity, which was increased when the AEMF was applied, indicating a potentially alternative therapy for the treatment of the periodontal disease.

Healthcare workers' hands as a vehicle for the transmission of virulent strains of Candida spp.: A virulence factor approach.

BACKGROUND: Although the majority of Candida infections are thought to come from endogenous sources, the healthcare workers' (HCWs) hands are being increasingly reported as vehicles for the transmission of pathogens. The aim of the present study was to evaluate the susceptibility of yeast isolated from the HCWs' hands and ICU (Intensive Care Unit) surfaces to antifungal agents and to determine the virulence potential and the genetic similarity between the same. METHODS: The susceptibility of yeasts from the HCWs' hands (n = 57) and ICU surfaces (n = 98) to conventional antifungals (fluconazole, voriconazole, amphotericin B and micafungin) was evaluated using the broth microdilution assay accordance with CLSI M27-A3. Additionally, some virulence factors such as adhesion and biofilm capacity on abiotic surfaces and on endothelial cells were evaluated, as well as germ tube formation. The similarity among yeast isolates were evaluated by the RAPD technique using the P4, OPA18 and OPE18 primers. RESULTS: Five species of Candida were found on the HCWs' hands (C. albicans, C. parapsilosis (sensu stricto), C. glabrata, C. tropicalis and C. krusei) and two on ICU surfaces (C. albicans and C. parapsilosis (sensu stricto)). The isolates from hands had higher resistance rates, with C. glabrata having the highest indices (100% FLU; 100% MFG). The similarity of C. albicans from HCWs and ICU surfaces was ≥80% according to the three primers analyzed. Candida spp. from hands had a greater potential for adhesion and biofilm formation on abiotic surfaces (p < 0.05). C. albicans from ICU surfaces had the greatest potential of adhesion on endothelial cells after 2 and 24 h, and presented high filamentation in SEM images and formed more and larger germ tubes (p < 0.05). CONCLUSION: the present study showed the significant virulence potential of yeasts transmitted in the hospital environment for the first time. Additionally, healthy people working in the ICU can carry these yeasts, which are capable of surviving in hospital surfaces, on their hands, offering a risk to patients, especially those who are immunocompromised.

Radiation used for head and neck cancer increases virulence in Candida tropicalis isolated from a cancer patient.

BACKGROUND: Studies have shown that radiation from radiotherapy increases the yeast colonization of patients. However it is not clear, if such radiation alters the yeast itself. The aim of the present study was therefore to report the direct impact of gamma radiation on Candida tropicalis. METHODS: C. tropicalis was obtained from a patient with a carcinoma, a suspension of this yeast containing 2.0 × 103 colony forming units per milliliter was prepared. It was submitted to gamma radiation dosage similar to that used in the treatment of head and neck cancer. After a cumulative dose of 7200 cGy some virulence attributes of C. tropicalis, including macro and micromorphological characteristics, adhesion and biofilm abilities, murine experimental infection and phagocytosis resistance were evaluated on irradiated and non-irradiated yeasts. RESULTS: After irradiation the colony morphology of the yeast was altered from a ring format to a smooth appearance in most colonies. Scanning electron microscopy revealed notable differences in the structures of both these colonies and the yeast cells, with the loss of pseudohyphae following irradiation and an increase in extracellular matrix production. The adherence and biofilm production of the yeast was greater following irradiation, both in terms of the number of yeasts and total biomass production on several abiotic surfaces and TR146 cells. The phagocytic index of the irradiated yeasts was not statistically different; however, the presence of cellular debris was detected in the kidneys of infected animals. Mice infected with irradiated yeasts developed an infection at the site of the yeast inoculation, although systemic infection was unchanged. CONCLUSIONS: Our findings show for the first time that C. tropicalis, one of the most important yeasts from colonization, which cause fatal candidemia in cancer patients, is affected by gamma irradiation, with changes to its virulence profile.

Emergence of Candida glabrata in vulvovaginal candidiasis should be attributed to selective pressure or virulence ability?

PURPOSE: Vulvovaginal candidiasis (VVC) is one of the most frequent female genital disorders and Candida glabrata is the second most common agent. Current study was aimed to study the susceptibility to antifungal agents of C. glabrata isolated from vaginal samples and some virulence attributes in order to better understand why this species is emerging as the main VVC agents. METHODS: A total of 60 C. glabrata vaginal isolates were included in this study. Firstly they were screened by susceptibility tests to antifungal agents. The isolates that showed sensitivity or resistance to fluconazole were evaluated for their virulence potential, including ability to adhere to polystyrene and vaginal ring, cell surface hydrophobicity (CSH) and capacity to form biofilm. RESULTS: Candida glabrata isolates varied significantly in adherence capacity, biofilm formation and CSH. However, it was possible to observe that isolates resistant to fluconazole adhered more efficiently to the vaginal ring and were statistically more able to form biofilm. CONCLUSION: These results allow hypothesizing that C. glabrata is an emergent agent in VVC probably because the treatment with fluconazole selects this species. But once adhered, yeasts remain on biotic or abiotic surfaces causing colonization or VVC symptomatology.

Overview of β-Glucans from Laminaria spp.: Immunomodulation Properties and Applications on Biologic Models.

Glucans are a group of glucose polymers that are found in bacteria, algae, fungi, and plants. While their properties are well known, their biochemical and solubility characteristics vary considerably, and glucans obtained from different sources can have different applications. Research has described the bioactivity of β-glucans extracted from the algae of the Laminaria genus, including in vivo and in vitro studies assessing pro- and anti-inflammatory cytokines, vaccine production, inhibition of cell proliferation, and anti- and pro-oxidant activity. Thus, the objective of this article was to review the potential application of β-glucans from Laminaria spp. in terms of their immunomodulatory properties, microorganism host interaction, anti-cancer activity and vaccine development.