Effect of the Pulsatilla decoction n-butanol extract on vulvovaginal candidiasis caused by Candida glabrata and on its virulence factors.

Vulvovaginal candidiasis (VVC) caused by Candida glabrata (C. glabrata) is more persistent and resistant to treatment than when caused by Candida albicans (C. albicans) and has been on the rise in recent years. The n-butanol extract of Pulsatilla Decoction (BEPD) has been shown to be effective in treating VVC caused by C. glabrata, but the underlying mechanism of action remains unclear. In this study, the experimenter conducted in vitro and in vivo experiments to explore the effects of BEPD on the virulence factors of C. glabrata, as well as its efficacy, with a focus on possible immunological mechanism in VVC caused by C. glabrata. The contents of Anemoside B4, Epiberberine, Berberine, Aesculin, Aesculetin, Phellodendrine and Jatrorrhizine in BEPD, detected by high-performance liquid chromatography, were 31,736.64, 13,529.66, 105,143.72, 19,406.20, 4952.67, 10,317.03, 2489.93 µg/g, respectively. In vitro experiments indicated that BEPD moderately inhibited the growth of C. glabrata, its adhesion, and biofilm formation, and affected the expression of efflux transporters in the biofilm state. In vivo experiments demonstrated that BEPD significantly reduced vaginal inflammatory manifestation and the release of proinflammatory cytokines and LDH in mice with VVC caused by C. glabrata. Moreover, it inhibited the Phosphorylation of EGFR, ERK, P38, P65, and C-Fos proteins. The results suggested that although BEPD moderately inhibits the growth and virulence factors of C. glabrata in vitro, it can significantly reduce vaginal inflammation by down-regulating the EGFR/MAPK signaling pathway in mice with VVC infected by C. glabrata.

Antifungal Efficacy of Antimicrobial Peptide Octominin II against Candida albicans.

Most clinically isolated Candida albicans strains are drug-resistant, emphasizing the urgent need to discover alternative therapies. In this study, the previously characterized Octominin was modified into a shorter peptide with an 18 amino acid sequence (1GWLIRGAIHAGKAIHGLI18) and named Octominin II. The secondary structure of Octominin II is a random coil with a helical turn and a positive charge (+2.46) with a hydrophobic ratio of 0.46. Octominin II inhibited C. albicans, C. auris, and C. glabrata with minimum inhibitory and fungicidal concentrations against C. albicans of 80 and 120 µg/mL, respectively. Field emission scanning electron microscopy confirmed that Octominin II treatment caused ultra-structural changes in C. albicans cells. Furthermore, membrane permeability results for the fluorescent indicator propidium iodide revealed modifications in cell wall integrity in Octominin II-treated C. albicans. Octominin II treatment increases the production of reactive oxygen species (ROS) in C. albicans. Gene expression studies revealed that Octominin II suppresses virulence genes of C. albicans such as CDR1, TUP1, AGE3, GSC1, SAP2, and SAP9. In addition, a nucleic acid binding assay revealed that Octominin II degraded genomic DNA and total RNA in a concentration-dependent manner. Additionally, Octominin II inhibited and eradicated C. albicans biofilm formation. Octominin II showed relatively less cytotoxicity on raw 264.7 cells (0-200 µg/mL) and hemolysis activity on murine erythrocytes (6.25-100 µg/mL). In vivo studies confirmed that Octominin II reduced the pathogenicity of C. albicans. Overall, the data suggests that Octominin II inhibits C. albicans by employing different modes of action and can be a promising candidate for controlling multidrug-resistant Candida infections.

Discovery of Pyrazolone Carbothioamide Derivatives as Inhibitors of the Pdr1-KIX Interaction for Combinational Treatment of Azole-Resistant Candidiasis.

Candida glabrata has emerged as an important opportunistic pathogen of invasive candidiasis due to increasing drug resistance. Targeting Pdr1-KIX interactions with small molecules represents a potential strategy for treating drug-resistant candidiasis. However, effective Pdr1-KIX inhibitors are rather limited, hindering the validation of target druggability. Here, new Pdr1-KIX inhibitors were designed and assayed. Particularly, compound B8 possessed a new chemical scaffold and exhibited potent KIX binding affinity, leading to enhanced synergistic efficacy with fluconazole to treat resistant C. glabrata infection (FICI = 0.28). Compound B8 acted by inhibiting the efflux pump and down-regulating resistance-associated genes through blocking the Pdr1-KIX interaction. Compound B8 exhibited excellent in vitro and in vivo antifungal potency in combination with fluconazole against azole-resistant C. glabrata. It also had direct antifungal effect to treat C. glabrata infection, suggesting new mechanisms of action independent of Pdr1-KIX inhibition. Therefore, compound B8 represents a promising lead compound for antifungal drug development.

Effect of effervescent tablets on removable partial denture hygiene.

PURPOSE: To evaluate the effectiveness of five alkaline peroxide-based effervescent tablets in reducing both biofilms and the food layer adhered on the cobalt-chromium surface. METHODS: Cobalt-chromium metal alloy specimens were contaminated with Candida albicans, Candida glabrata, Streptococcus mutans and Staphylococcus aureus. After biofilm maturation, the specimens were immersed in Polident 3 Minute, Polident for Partials, Efferdent, Steradent, Corega Tabs or distilled water (control). Residual biofilm rates were determined by colony forming units counts and biofilm biomass. In parallel, to investigate the denture cleaning capability of effervescent tablets, artificially contaminated removable partial dentures were treated with each cleanser. Data were analyzed by Kruskal-Wallis followed by Dunn post hoc test or ANOVA followed by Tukey post hoc test (α= 0.05). RESULTS: None of the hygiene solutions reduced C. albicans biofilm. Efferdent and Corega Tabs promoted reduction of C. glabrata biofilm, while Steradent was favorable against S. aureus biofilm. For S. mutans, lower biofilm rates were observed after immersion in Polident for Partials and Steradent. The effervescent tablets showed good cleaning performance, removing an artificial layer with carbohydrates, proteins, and fats, however, they were not effective in removing aggregated mature biofilm. CLINICAL SIGNIFICANCE: The different effervescent tablets presented favorable antimicrobial activity against C. glabrata, S. mutans and S. aureus on cobalt-chromium surfaces and showed cleaning capability. However, for an appropriate biofilm control, a complementary method should be evaluated since none of the peroxide-based solutions reduced C. albicans biofilms or substantially removed aggregated biofilm.

Metabolite profiling, antifungal, biofilm formation prevention and disruption of mature biofilm activities of Erythrina senegalensis stem bark extract against Candida albicans and Candida glabrata.

The antifungal activity of the 70% ethanol stem bark extract of Erythrina senegalensis (ESB) against different strains and drug resistant clinical isolates of Candida albicans and Candida glabrata were evaluated in the study. The effect of ESB on biofilms as well as its activity in combination with fluconazole, nystatin or caspofungin against the Candida strains were also evaluated. We then evaluated the antifungal activity of a microemulsion formulation of ESB against planktonic and biofilms of the Candida species. UPLC-QTOF-MS2 analysis was then undertaken to identify the phytoconstituents of the extract and UPLC fingerprints developed for the routine authentication as part of quality control measures. ESB exerted strong antifungal activities against C. albicans ATCC 10231 and SC5314 strains, and C. glabrata ATCC 2001 strain with minimum inhibitory concentration (MIC) values from 3.91 to 31.25 µg/mL and minimum fungicidal concentrations (MFCs) that ranged from 62.5 to 250 µg/mL. It also exhibited potent antifungal activities (MIC = 4-64 µg/mL) against a collection of C. albicans and C. glabrata clinical isolates that were resistant to either nystatin or azole antifungals. The formulated ESB demonstrated higher antifungal potency against the C. albicans and C. glabrata strains with MIC values of 3.91-31.25 µg/mL which was the same as the MFC values. The extract and its microemulsion formulation were active against biofilms of the strains of the Candida species inhibiting their biofilm formations (SMIC50 = 16-64 µg/mL) and their preformed biofilms (SMIC50 = 128 ->512 µg/mL). ESB also exhibited synergistic antifungal action with fluconazole and nystatin against C. albicans ATCC 10231 and C. glabrata ATCC 2001 strains in the checkerboard assay. Chemical characterization of the extract revealed the presence of phenolic compounds such as flavonoids and their prenylated derivatives, anthracene glycosides and alkaloids. UPLC Fingerprints of the extract was also developed and validated for routine identification and authentication of the stem bark of E. senegalensis. The study findings have demonstrated that the stem bark of E. senegalensis is as a potential source of bioactive compounds that could be developed as novel antifungal agents.

Evaluation of in vitro antifungal effects of synthetic and herbal mouth rinses on oral Candida albicans and Candida glabrata.

Mouth rinses which function as breath fresheners, medicaments, and antiseptics can also deliver oral therapeutic agents. This study evaluated and compared the antifungal effects of synthetic and herbal mouth rinses on oral C. albicans and C. glabrata via disk diffusion, minimal inhibition concentration (MIC), minimum fungicidal concentration (MFC), time-kill assay, and growth profile tests. The four chemical mouth rinses, namely Brand O (A), Brand M (B), Brand H (C), and Brand B (D) used in the study showed positive antifungal activity in these two species. The average diameter of the inhibition zones obtained from the disk diffusion test was higher in mouth rinse B (C. albicans = 12.0 ± 0.9 mm, C. glabrata = 13.5 ± 0.8 mm) compared to those in C, A and D. Both Candida species exhibited similar MIC and MFC values, ranging from 1.63 ± 0.5 to 18.75 ± 0.0 µg/mL and 6.51 ± 2.01 to 50.00 ± 9.36 µg/mL, respectively. These synthetic mouth rinses had efficient killing activity eliminating 50% of the growing population of both Candida spp. following 15 seconds exposure time. Analyses of the growth profile curves showed that mouth rinses B and A resulted in rapid growth depletion of both Candida spp. Meanwhile, three herbal mouth rinses, namely Brand S (E), Brand C (F), and Brand P (G), were less effective against C. albicans and C. glabrata. Mouth rinses B and A contained cetylpyridinium chloride and chlorhexidine, respectively, and could be an effective alternative for controlling and preventing oral candidiasis.

Characterization of nanomaterials synthesized from Spirulina platensis extract and their potential antifungal activity.

Nowadays, fungal infections increase, and the demand of novel antifungal agents is constantly rising. In the present study, silver, titanium dioxide, cobalt (II) hydroxide and cobalt (II,III) oxide nanomaterials have been synthesized from Spirulina platensis extract. The synthesis mechanism has been studied using GCMS and FTIR thus confirming the involvement of secondary metabolites, mainly amines. The obtained products have been analysed using XRD, SEM, TGA and zeta potential techniques. The findings revealed average crystallite size of 15.22 nm with 9.72 nm for oval-shaped silver nanoparticles increasing to 26.01 nm and 24.86 nm after calcination and 4.81 nm for spherical-shaped titanium dioxide nanoparticles which decreased to 4.62 nm after calcination. Nanoflake shape has been observed for cobalt hydroxide nanomaterials and for cobalt (II, III) oxide with crystallite size of 3.52 nm and 13.28 nm, respectively. Silver nanoparticles showed the best thermal and water dispersion stability of all the prepared structures. Once subjected to three different Candida species (C. albicans, C. glabrata, and C. krusei) silver nanoparticles and cobalt (II) hydroxide nanomaterials showed strong antifungal activity at 50 µg/mL with minimum inhibitory concentration (MIC) values. After light exposition, MIC values for nanomaterials decreased (to 12.5 µg/mL) for C. krusei and increased (100 µg/mL) for C. albicans and C. glabrata.

Characterization of Cervus timorensis velvet antler and its effect on biofilm formation of Candida species.

Oral biofilms comprise extracellular polysaccharides and polymicrobial microorganisms. The objectives of the study were to characterize the deer velvet antler (DVA) compounds and their effect on Candida species biofilm formation with the hypothesis that DVA inhibits the biofilm of Candida spp. Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry (LC-QTOF-MS) was conducted to characterize the DVA compounds. To study the effect of DVA on biofilm, Candida albicans ATCC MYA-4901 (ALT5), AIDS isolate (ALC2), oral cancer isolate (ALC3), C. dubliniensis ATCC MYA-2975, C. glabrata ATCC 90030, C. krusei 14 243, C. lusitaniae ATCC 34449, C. parapsilosis ATCC 22019, and C. tropicalis ATCC 13803 were inoculated with DVA in separate wells of a 96-well plate containing RPMI-1640 followed by 72 h incubation. A total of 45 compounds were detected in the DVA extract. C. lusitaniae exhibited a higher percentage of biofilm biomass reduction when treated with DVA extract (66.10% ± 5.33), followed by ALC3 (44.12% ± 6.24). However, C. glabrata, C. krusei, and C. parapsilosis showed no reduction in biofilm biomass after being treated with DVA extract. Most Candida strains also exhibited decreased total cell count when treated with DVA extract, except for ALC3 and C. krusei. ALT5 had the lowest total cell count (0.17 × 105 cells/ml) when cultured with DVA extract. In conclusion, DVA extract inhibits Candida spp. biofilm formation except for C. glabrata, C. krusei, and C. parapsilosis.

The study determines deer velvet antler (DVA) compounds and their effect on Candida species biofilm formation. A total of 45 compounds were detected in the DVA extract. Most Candida spp. exhibited a higher percentage of biofilm reduction and decreased total cell count when treated with DVA extract.

Candida glabrata - basic characteristics, virulence, treatment, and resistance.

Fungal infections are currently a serious health concern. Life-threatening conditions that occur mainly in immunocompromised patients are largely caused by representatives of the genus Candida. The most common causative agent is the yeast Candida albicans, but in recent years there has been a significant shift towards Candida glabrata and other so-called non-albicans Candida yeasts (e.g. Candida tropicalis or Candida parapsilosis). Invasive infections caused by the multidrug-resistant yeast Candida auris are associated with high mortality. There are several differences between C. glabrata and other causative agents of candidiasis in biological characteristics and virulence factors. The innate increased resistance to azoles along with the ability to rapidly acquire resistance to other groups of antifungal agents is a dangerous combination which makes it difficult to manage Candida infections. A better understanding of the virulence factors and mechanisms of resistance to antifungal agents can benefit the management of Candida infections. Equally important is the search for new target sites for antifungal therapy. The present work briefly summarizes the existing knowledge in this area.

Comparison of simulated candidemia detection during prophylactic antifungal therapy.

BACKGROUND: We investigated the neutralization performance of various automated blood culture systems for antifungal agents with regard to the most commonly isolated Candida species. METHODS: In this study, we evaluated the time to detection (TTD) of simulated candidemia for 6 Candida spp. (C. albicans, C. auris, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis) in 3 automated blood culture systems (BACTEC™ FX, BACT/ALERT® 3D, and BACT/ALERT® VIRTUO®), with or without trough and peak levels of eight antifungal agents (amphotericin B, anidulafungin, caspofungin, fluconazole, itraconazole, micafungin, posaconazole, and voriconazole). RESULTS: Caspofungin and micafungin significantly prolonged the TTDs for most of the tested strains in the 3 blood culture instruments, especially at peak concentrations. CONCLUSION: Peak concentrations of caspofungin and micafungin influence the performance of blood culture detection systems. Therefore, one should be careful about the possibility of prolonged TTDs for candidemia when using the abovementioned antifungal agents.

Investigation of the Defective Growth Pattern and Multidrug Resistance in a Clinical Isolate of Candida glabrata Using Whole-Genome Sequencing and Computational Biology Applications.

Candida glabrata is increasingly isolated from blood cultures, and multidrug-resistant isolates have important implications for therapy. This study describes a cholesterol-dependent clinical C. glabrata isolate (ML72254) that did not grow without blood (containing cholesterol) on routine mycological media and that showed azole and amphotericin B (AmB) resistance. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and whole-genome sequencing (WGS) were used for species identification. A modified Etest method (Mueller-Hinton agar supplemented with 5% sheep blood) was used for antifungal susceptibility testing. WGS data were processed via the Galaxy platform, and the genomic variations of ML72254 were retrieved. A computational biology workflow utilizing web-based applications (PROVEAN, AlphaFold Colab, and Missense3D) was constructed to predict possible deleterious effects of these missense variations on protein functions. The predictive ability of this workflow was tested with previously reported missense variations in ergosterol synthesis genes of C. glabrata. ML72254 was identified as C. glabrata sensu stricto with MALDI-TOF, and WGS confirmed this identification. The MICs of fluconazole, voriconazole, and amphotericin B were >256, >32, and >32 µg/mL, respectively. A novel frameshift mutation in the ERG1 gene (Pro314fs) and many missense variations were detected in the ergosterol synthesis genes. None of the missense variations in the ML72254 ergosterol synthesis genes were deleterious, and the Pro314fs mutation was identified as the causative molecular change for a cholesterol-dependent and multidrug-resistant phenotype. This study verified that web-based computational biology solutions can be powerful tools for examining the possible impacts of missense mutations in C. glabrata. IMPORTANCE In this study, a cholesterol-dependent C. glabrata clinical isolate that confers azole and AmB resistance was investigated using artificial intelligence (AI) technologies and cloud computing applications. This is the first of the known cholesterol-dependent C. glabrata isolate to be found in Turkey. Cholesterol-dependent C. glabrata isolates are rarely isolated in clinical samples; they can easily be overlooked during routine laboratory procedures. Microbiologists therefore need to be alert when discrepancies occur between microscopic examination and growth on routine media. In addition, because these isolates confer antifungal resistance, patient management requires extra care.

Bioactive compounds with antifungal activity against pathogens isolated from pregnant woman: Gallesia integrifolia (garlic wood) is a promising treatment for vulvovaginal candidiasis.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves, bark, and roots of Gallesia integrifolia are consumed in folk medicine through infusion, decoction, and topical preparation by crushing because of its pharmacological properties in several peripheral system disorders, including microbial infections. The presence of various molecules in different parts of the plant likely confers this species' fungicidal action, but scientific evidence is lacking. Vulvovaginal candidiasis mainly affects women of reproductive age. When left untreated, it can cause pregnancy complications. Currently available antifungals often cause undesirable side effects. New alternative therapeutic strategies based on medicinal plants have been proposed. AIM: To investigate the antifungal activity of G. integrifolia against vulvovaginal candidiasis secretion in pregnant women. MATERIALS AND METHODS: Antifungal activity was determined by the minimum inhibitory concentration (MIC), determined by broth microdilution method using Candida spp (NEWP1210), C. albicans (CCCD-CC001), C. tropicalis (CCCD-CC002) standard and clinical isolates from pregnant women with vulvovaginal candidiasis. Nystatin and fluconazole were used as positive controls. The chemical composition of essential oils that were extracted from leaves, flowers, and fruits of G. integrifolia was determined by gas chromatography coupled to mass spectrometry. Reverse docking was used to suggest a possible target in Candida. Conventional docking was used to identify the most probable compound that inhibits fungal growth. RESULTS: A total of 24 compounds were identified, accounting for ∼99% of volatile constituents in the essential oils. Leaves of G. integrifolia contained 3,5-dithiahexanol-5,5-dioxide (40.93%), flowers contained methionine ethyl ester (46.78%), and fruits contained 2,8-dithianonane (54.01%) as the most abundant compounds. The MICs of essential oils of leaves, flowers, and fruits of G. integrifolia against standard strains of Candida spp, C. albicans, and C. tropicalis ranged from 13.01 to 625.00 µg/mL. The essential oil of flowers more effectively inhibited Candida spp. Essential oils of leaves and flowers were similar to fluconazole against C. albicans. Essential oils of flowers and fruits were similar to fluconazole against C. tropocalis. In Candida yeast species that were isolated from vaginal secretion samples from pregnant patients, the MICs of leaves and flowers ranged from 52.08 to 5000.00 µg/mL. The essential oil of leaves (277.77 µg/mL) was the most active against C. albicans. No significant differences were found between the essential oils of leaves and flowers against C. glabrata. Docking simulations suggested that phytol in leaves and flowers was responsible for the antimicrobial effect. CONCLUSION: The present results suggest the potential therapeutic use of G. integrifolia, especially its leaves and flowers, against Candida and vulvovaginal candidiasis.

A comparative study of in-vitro and in-silico anti-candidal activity and GC-MS profiles of snow mountain garlic vs. normal garlic.

AIM: The study aimed to profile the volatile phytocomposition of snow mountain garlic (SMG) compared to normal garlic and investigate the anti-Candida efficacy against clinically relevant multi-drug resistant isolates of Candida species. METHODS AND RESULTS: Herein, SMG has shown significantly superior fungicidal power at 2x-MIC dose against C. albicans and C. glabrata in killing kinetic evaluation unlike the fungistatic effect of normal garlic. GC-MS headspace-based profiling of SMG showed 5 unique volatile compounds and a 5-fold higher content of saponins than normal garlic. In an in-silico analysis, cholesta-4,6-dien-3-ol,(3-beta) was uniquely identified in SMG as a potential inhibitor with high binding affinity to the active site of exo-1,3-betaglucan synthase, an established anti-candida drug target crucial for the biofilm matrix formation, thus suggesting a plausible anti-Candida mechanism. CONCLUSION: The in-vitro and in-silico studies have demonstrated the Candida-cidal and anti-biofilm activities of SMG, distinguishing it from the Candida-static efficacy of normal garlic. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that identifies several phytochemical signatures of SMG along with a potential anti-Candida compound, that is cholesta-4,6-dien-3-ol,(3-beta)-, which appears worthy of detailed studies in the future to explore the utility of SMG as a fungal phytotherapy agent, especially against drug-resistant Candida sp.

Antibacterial and Antifungal Efficacy of Medium and Low Weight Chitosan-Shelled Nanodroplets for the Treatment of Infected Chronic Wounds.

Purpose: Medium versus low weight (MW vs LW) chitosan-shelled oxygen-loaded nanodroplets (cOLNDs) and oxygen-free nanodroplets (cOFNDs) were comparatively challenged for biocompatibility on human keratinocytes, for antimicrobial activity against four common infectious agents of chronic wounds (CWs) - methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Candida albicans and C. glabrata - and for their physical interaction with cell walls/membranes. Methods: cNDs were characterized for morphology and physico-chemical properties by microscopy and dynamic light scattering. In vitro oxygen release from cOLNDs was measured through an oximeter. ND biocompatibility and ability to promote wound healing in human normoxic/hypoxic skin cells were challenged by LDH and MTT assays using keratinocytes. ND antimicrobial activity was investigated by monitoring upon incubation with/without MW or LW cOLNDs/cOFNDs either bacteria or yeast growth over time. The mechanical interaction between NDs and microorganisms was also assessed by confocal microscopy. Results: LW cNDs appeared less toxic to keratinocytes than MW cNDs. Based on cell counts, either MW or LW cOLNDs and cOFNDs displayed long-term antimicrobial efficacy against S. pyogenes, C. albicans, and C. glabrata (up to 24 h), whereas a short-term cytostatic effects against MRSA (up to 6 h) was revealed. The internalization of all ND formulations by all four microorganisms, already after 3 h of incubation, was showed, with the only exception to MW cOLNDs/cOFNDs that adhered to MRSA walls without being internalized even after 24 h. Conclusion: cNDs exerted bacteriostatic and fungistatic effects, due to the presence of chitosan in the outer shell and independently of oxygen addition in the inner core. The duration of such effects strictly depends on the characteristics of each microbial species, and not on the molecular weight of chitosan in ND shells. However, LW chitosan was better tolerated by human keratinocytes than MW. For these reasons, the use of LW NDs should be recommended in future research to assess cOLND efficacy for the treatment of infected CWs.

CRISPR-Cas9 approach confirms Calcineurin-responsive zinc finger 1 (Crz1) transcription factor as a promising therapeutic target in echinocandin-resistant Candida glabrata.

Invasive fungal infections, which kill more than 1.6 million patients each year worldwide, are difficult to treat due to the limited number of antifungal drugs (azoles, echinocandins, and polyenes) and the emergence of antifungal resistance. The transcription factor Crz1, a key regulator of cellular stress responses and virulence, is an attractive therapeutic target because this protein is absent in human cells. Here, we used a CRISPR-Cas9 approach to generate isogenic crz1Δ strains in two clinical isolates of caspofungin-resistant C. glabrata to analyze the role of this transcription factor in susceptibility to echinocandins, stress tolerance, biofilm formation, and pathogenicity in both non-vertebrate (Galleria mellonella) and vertebrate (mice) models of candidiasis. In these clinical isolates, CRZ1 disruption restores the susceptibility to echinocandins in both in vitro and in vivo models, and affects their oxidative stress response, biofilm formation, cell size, and pathogenicity. These results strongly suggest that Crz1 inhibitors may play an important role in the development of novel therapeutic agents against fungal infections considering the emergence of antifungal resistance and the low number of available antifungal drugs.

Sodium houttuyfonate enhances the mono-therapy of fluconazole on oropharyngeal candidiasis (OPC) through HIF-1α/IL-17 axis by inhibiting cAMP mediated filamentation in Candida albicans-Candida glabrata dual biofilms.

Candida albicans and Candida glabrata are two common opportunistic fungi that can be co-isolated in oropharyngeal candidiasis (OPC). Hypha is a hallmark of the biofilm formation of C. albicans, indispensable for the attachment of C. glabrata, which is seldom in mycelial morphology. Increasing evidence reveals a hypoxic microenvironment in interior fungal biofilms, reminding of a fact that inflammation is usually accompanied by oxygen deprivation. As a result, it is assumed that the disaggregation of hypha-mediated hypoxia of biofilms might be a solution to alleviate OPC. Based on this hypothesis, sodium houttuyfonate (SH), a well-identified traditional herbal compound with antifungal activity, is used in combination with fluconazole (FLU), a well-informed synthesized antimycotics, to investigate their impact on filamentation in C. albicans and C. glabrata dual biofilms and the underlying mechanism of their combined treatment on OPC. The results show that compared with the single therapy, SH plus FLU can inhibit the hyphal growth in the mixed biofilms in vitro, decrease the fungal burden of oral tissues and internal organs, restore mucosal epithelial integrity and function, and reduce hypoxic microenvironment and inflammation in a mice OPC model. The possible mechanism of the combined therapy of SH plus FLU can be attributed to the regulation of HIF-1α/IL-17A axis through direct abrogation of the dual Candida biofilm formation. This study highlights the role of HIF-1α/IL-17A axis and the promising application of SH as a sensitizer of conventional antifungals in the treatment of OPC.

Green synthesis of selenium nanoparticles and evaluate their effect on the expression of ERG3, ERG11 and FKS1 antifungal resistance genes in Candida albicans and Candida glabrata.

Drug resistance in Candida species has been considerably increased in the last decades. Given the opposition to antifungal agents, toxicity and interactions of the antimicrobial drugs, identifying new antifungal agents seems essential. This study assessed the antifungal effects of nanoparticles (NPs) on the standard strains of Candida albicans and Candida glabrata and determined the expression genes, including ERG3, ERG11 and FKS1. Selenium nanoparticles (Se-NPs) were biosynthesized with a standard strain of C. albicans and approved by several methods including, ultraviolet-visible spectrophotometer, X-ray diffraction technique, Fourier-transform infrared analysis, field-emission scanning electron microscopy and EDX diagram. The antifungal susceptibility testing performed the minimum inhibitory concentrations (MICs) using the CLSI M27-A3 and M27-S4 broth microdilution method. The expression of the desired genes was examined by the real-time PCR assay between untreated and treated by antifungal drugs and Se-NPs. The MICs of itraconazole, amphotericin B and anidulafungin against C. albicans and C. glabrata were 64, 16 and 4 µg ml-1 . In comparison, reduced the MIC values for samples treated with Se-NPs to 1 and 0·5 µg ml-1 . The results obtained from real-time PCR and analysis of the ∆∆Cq values showed that the expression of ERG3, ERG11 and FKS1 genes was significantly down-regulated in Se-NPs concentrations (P < 0·05). This study's evidence implies biosafety Se-NPs have favourable effects on the reducing expression of ERG3, ERG11 and FKS1 antifungal resistance genes in C. albicans and C. glabrata.

Ent-hardwickiic acid from C. pubiflora and its microbial metabolites are more potent than fluconazole in vitro against Candida glabrata.

The incidence of Candida glabrata infections has rapidly grown and this species is among those responsible for causing invasive candidiasis with a high mortality rate. The diterpene ent-hardwickiic acid is a major constituent in Copaifera pubiflora oleoresin and the ethnopharmacological uses of this oleoresin by people from Brazilian Amazonian region point to a potential use of this major constituent as an antimicrobial. Therefore, the goal of this study was to evaluate the antifungal activity of ent-hardwickiic acid against Candida species and to produce derivatives of this diterpene by using microbial models for simulating the mammalian metabolism. The microbial transformations of ent-hardwickiic acid were carried out by Aspergillus brasiliensis and Cunninghamella elegans and hydroxylated metabolites were isolated and their chemical structures were determined. The antifungal activity of ent-hardwickiic acid and its metabolites was assessed by using the microdilution broth method in 96-well microplates and compared with that of fluconazole. All the diterpenes showed fungistatic effects (ranging from 19·7 to 75·2 µmol l-1 ) against C. glabrata at lower concentrations than fluconazole (163·2 µmol l-1 ) and were more potent fungicides (ranging from 39·5 to 150·4 µmol l-1 ) than fluconazole, which showed fungicidal effect at the concentration of 326·5 µmol l-1 .

Influence of glucose supplementation on biofilm formation of Candida albicans and Candida glabrata isolated from diabetic and non-diabetic individuals.

OBJECTIVE: This study evaluated the effect of different glucose concentration on biofilm formation of Candida albicans and Candida glabrata strains isolated from diabetic and non-diabetic individuals. METHODS: The study was divided into two stages: (I) selection and identification of 48 C. albicans and C. glabrata strains by polymerase chain reaction followed by restriction fragment length polymorphism analysis (PCR/RFLP); (II) evaluation of biofilm formation by means of viability rates (colony-forming units), biofilm dry matter (mg) and biofilm-covered areas (µm2). Statistical comparisons were performed through nonparametric analysis of longitudinal data in factorial experiments with pairwise comparisons using Friedman Conover's test (α = 0.05). RESULTS: All the Candida spp. had their identifications confirmed by PCR/RFLP. C. albicans biofilm of strains from diabetic individuals cultivated in different glucose concentration showed higher viability rates than strains from non-diabetic individuals. No difference was observed on viability of C. glabrata biofilm. Regarding biofilm dry matter, C. albicans biofilm of strains from diabetic individuals cultivated in different glucose concentration showed lower amount in weight than strains from non-diabetic individuals. In C. glabrata strains, this result was only observed in biofilms cultivated with no glucose supplementation. With regard to biofilm-covered areas, only glucose supplementation and non-diabetic condition showed a positive effect on C. albicans biofilm development, and no condition affected C. glabrata biofilm formation. CONCLUSION: The strain type (C. albicans and C. glabrata) isolated from diabetic and non-diabetic individuals influenced on biofilm formation, but glucose supplementation did not.

Avaliação in vitro de formulações do óleo essencial de citronela (Cymbopogon nardus) sobre propriedades biológicas, físicas e mecânicas de resina acrílica para prótese dentária / In vitro evaluation of citronella (Cymbopogon nardus) essential oil formulations on biological, physical and mechanical properties of acrylic resin for dental prosthesis

O controle de formação de biofilme deve ser promovido por meio de técnicas diárias de higienização, sendo uma delas o uso de soluções de desinfecção. Dentre as soluções químicas comercialmente disponíveis, algumas podem ocasionar efeitos adversos quando utilizadas em longo prazo. Dessa forma, a busca por métodos alternativos de desinfecção, com soluções que não alterem as propriedades do material e que sejam inertes para o seu usuário torna-se essencial. Este estudo teve como objetivos: avaliar in vitro, a ação antifúngica de formulações de 5 mg/mL à base do óleo de citronela em biofilmes mistos de espécie de Candida em resina acrílica ativada termicamente (RAAT) para base protética por meio de contagem do número de unidades formadoras de colônias (UFC/mL), em dois tempos de exposição (15 e 30 minutos) e avaliar a citotoxicidade das formulações em células da linhagem epitelial HaCat, e verificar a ação destas formulações de citronela a 5 mg/mL na alteração de propriedades físicas e mecânicas de amostras de RAAT. Inicialmente, foram feitas as formulações à base de citronela em forma de emulsão e nanoemulsão. Em seguida, por meio de ensaios de microdiluição em caldo, foi obtida a concentração inibitória mínima (CIM) e concentração fungicida mínima (CFM) das cepas de Candida testadas. Foram confeccionadas amostras de RAAT para a realização dos ensaios, sendo 96 amostras para o ensaio de biofilme, divididas de acordo com os grupos: GI - controle negativo (Biofilme misto C. albicans ATCC + C. glabrata ATCC + C. albicans oral 6892 + C. albicans oral 7037); GII- Nanoemulsão de Citronela 5mg/mL; GIIIControle da Nanoemulsão (Tween 20); GIV- Emulsão de Citronela 5mg/mL; GV- Controle da Emulsão (Goma Xantana); GVI- Clorexidina 0,12% (Periogard), e 20 para o ensaio de viabilidade celular, divididas nos grupos: GI- Nanoemulsão de Citronela 5mg/mL; GII- Controle da Nanoemulsão (Tween 20); GIII- Emulsão de Citronela 5mg/mL; GIV- Controle da Emulsão (Goma Xantana); GV- Clorexidina 0,12% (Periogard). Biofilmes mistos de Candida foram formados sobre as superfícies das amostras, e foram quantificados por meio de contagem de unidades formadoras de colônias (UFCs). Para o teste de viabilidade celular utilizou-se células epiteliais da linhagem HaCaT, avaliadas através da coloração com resazurina. Para avaliação das propriedades físicas e mecânicas foram confeccionadas 50 amostras retangulares e 50 circulares de RAAT, separadas aleatoriamente em 5 grupos, de acordo com a formulação utilizada: GI ­ controle (água destilada); GII ­ saliva artificial; GIII ­ nanoemulsão de citronela a 5mg/mL; GIV - emulsão de citronela a 5mg/mL e GV - Clorexidina 0,12% (Periogard). As amostras foram imersas nas respectivas formulações, simulando a desinfecção de próteses dentarias pelo período de 30 minutos diários, durante 6 meses. Após este período, foi feita a análise da alteração de rugosidade de superfície (Ra - µm), estabilidade de cor (CIEDE 2000), avaliação da microdureza Knoop e resistência a flexão (MPa) das amostras. Os dados obtidos foram submetidos às análises estatísticas (p< 0,05). As formulações fitoterápicas à base de citronela apresentaram valores de CIM e CFM que variaram entre 0,156-1 mg/mL sobre as espécies de Candida e inibiram o crescimento do biofilme misto, apresentando uma redução estatisticamente significativa na contagem de UFC/mL, de até 2,7 Log destes fungos, principalmente quando submetidas ao tempo de exposição de 30 minutos. As formulações fitoterápicas testadas não apresentaram diferenças estatísticas entre si, apresentando viabilidade da célula HaCaT maior que 70%, diferentemente da clorexidina a 0,12%. Não houve diferença nos valores médios de rugosidade de superfície em nenhum dos grupos após serem submetidos à imersão nas formulações testes, com exceção do grupo controle, que apresentou diferença estatística significativa na rugosidade de superfície após o período de imersão. Nos valores de alteração de cor (ΔE00) foi possível observar que não houve diferença estatística significativa entre os grupos experimentais antes e após serem submetidos a imersão nas formulações testes. Nos valores de microdureza Knoop houve diferença estatística significativa apenas entre o grupo controle e o grupo emulsão de citronela. Para os dados de resistência flexural, módulo de elasticidade, força máxima e alongamento não houve diferença estatística significativa entre os grupos após imersão. Em suma, concluiu-se que as formulações fitoterápicas à base de citronela apresentaram efeito antifúngico sobre as diferentes espécies de Candida em superfície de resina acrílica para próteses dentárias, não apresentaram efeito citotóxico sobre células da linhagem epitelial, e se mostraram seguras como formulações antissépticas no que se refere às propriedades físicas e mecânicas estudadas, sendo potencialmente promissoras para serem indicadas como soluções desinfetantes para próteses dentárias(AU)

The control of biofilm formation should be promoted through daily hygiene techniques such as the use of disinfection solutions. Among the commercially available chemical solutions, some can lead to adverse effects in long-term use. Thus, the search for alternative methods of disinfection with solutions that do not interfere with properties of the material and those inert to the user becomes essential. This study aimed to: evaluate, in vitro, the antifungal effect of 5 mg/mL formulations based on citronella oil on mixed biofilms of Candida species in thermally activated acrylic resin (TAAR) for prosthetic base by counting the number of colony forming units (CFU/mL), in two exposure times (15 and 30 minutes) and to evaluate the cytotoxicity of the formulations in cells of the HaCat epithelial lineage, and to verify the action of these formulations of citronella at 5 mg/mL in the alteration of physical and mechanical properties of TAAR samples. Initially, citronella-based formulations were prepared as emulsion and nanoemulsion. Then, by means of broth microdilution assays, the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the tested Candida strains were obtained. For the biofilm test, 96 TAAR samples were prepared and assigned to the groups, as follows: GI - negative control (Mixed biofilm C. albicans ATCC + C. glabrata ATCC + C. albicans oral 6892 + C. albicans oral 7037); GII- Citronella Nanoemulsion 5mg/mL; GIII- Nanoemulsion Control (Tween 20); GIV- Citronella Emulsion 5mg/mL; GVEmulsion Control (Xanthan Gum); GVI- Chlorhexidine 0,12% (Periogard). For the cell viability assay, 20 samples were prepared and assigned to the following groups: GI- Citronella Nanoemulsion 5mg/mL; GII- Nanoemulsion Control (Tween 20); GIII- Citronella Emulsion 5mg/mL; GIV- Emulsion Control (Xanthan Gum); GV- Chlorhexidine 0,12% (Periogard). Mixed Candida biofilms were formed on the surfaces of the samples, and were quantified by counting colony forming units (CFUs). For the cell viability test, epithelial cells of the HaCaT lineage were used, evaluated by staining with resazurin. To evaluate the physical and mechanical properties, 50 rectangular and 50 circular samples of TAAR were prepared and randomly assigned into 5 groups, according to the formulation used: GI ­ control (distilled water); GII ­ artificial saliva; GIII ­ citronella nanoemulsion at 5mg/mL; GIV ­ citronella emulsion at 5mg/mL and GV ­ chlorhexidine 0,12% (Periogard). The samples were immersed in the respective formulations, simulating the disinfection of dental prostheses for a period of 30 minutes daily, for 6 months. After this period, the analysis of surface roughness change (Ra - µm), color stability (CIEDE 2000), evaluation of Knoop microhardness and flexural strength (MPa) of the samples was performed. The data obtained were submitted to statistical analysis (p< 0,05). The phytotherapic formulations based on citronella presented MIC and MFC values that varied between 0,156-1 mg/mL on Candida species and inhibited the growth of mixed biofilm, showing a statistically significant reduction in the CFU/mL counts, up to 2,7 Log of these fungi, mainly when subjected to an exposure time of 30 minutes. The phytotherapic formulations tested did not show statistical differences between them, showing HaCaT cell viability greater than 70%, unlike 0,12% chlorhexidine. There was no difference in the mean values of surface roughness in any of the groups after being subjected to immersion in the test formulations, except for the control group, that presented a statistically significant difference in surface roughness after the immersion period. Regarding color change (ΔE00) it was possible to observe that there was no statistically significant difference between the experimental groups before and after being subjected to immersion in the test formulations. In the Knoop microhardness values, there was a statistically significant difference only between the control group and the citronella emulsion group. For data on flexural strength, modulus of elasticity, maximum force and stretching, there was no statistically significant difference between the groups after immersion. In summary, it can be concluded that phytotherapic formulations based on citronella had an antifungal effect on different Candida species on acrylic resin surfaces for dental prostheses, besides not presenting a cytotoxic effect on cells of the epithelial lineage, and proved to be safe as antiseptic formulations in the which refers to the physical and mechanical properties studied, being potentially promising to be indicated as disinfectant solutions for dental prostheses(AU)