Candida Biofilms Research Articles

The inhibitory effects of tyrosol on clinical Candida glabrata planktonic and biofilm cells

Biofilm formation is an important problem in the healthcare industry and veterinary medicine and is very common in natural, industrial or hospital environments. Microorganisms can become very resistant to antimicrobials and environmental factors by biofilm forming on biotic or abiotic surfaces. There is a need to develop new, effective and specific antimicrobials that can reduce pathogenicity in biofilm formation that threatens public health due to their role in medical device-related or infectious diseases. Candida species are opportunistic pathogenic yeasts and can cause superficial or disseminated infections. Especially C. glabrata is one of the most common microorganisms causing fungal infections in immunocompromised patients and drug resistance is observed when associated with biofilm. Tyrosol (2-[4-hydroxyphenyl] ethanol) can act as both a quorum sensing molecule and an exogenous agent on Candida species. In this study, the antifungal activity of tyrosol against a clinical C. glabrata isolate was investigated on both planktonic and biofilm forms. Broth microdilution test results demonstrated the inhibitory effect of tyrosol on C. glabrata. Transmission electron microscopic findings showed that tyrosol affected the planktonic C. glabrata cells in a multi targeted manner, and in the groups treated with tyrosol, significant damage was observed in the cell wall, cell membrane, cytoplasm, nucleus and mitochondria. Also, scanning electron microscopic images confirmed biofilm reduction in pre-/post-biofilm applications as a result of tyrosol treatment. In conclusion, tyrosol may be a potential alternative candidate for reducing the C. glabrata biofilm.

In Vitro Study on Green Propolis as a Potential Ingredient of Oral Health Care Products.

Propolis is increasingly being discussed as an alternative to commonly used antiseptics. This in vitro study focused on the ethanolic extract of green Brazilian propolis (EEPg) as an additive in an oral health care product. We investigated (i) a potential inflammation-modulation activity of EEPg when a periodontal or Candida biofilm was exposed to monocytic (MONO-MAC-6) cells, (ii) the adhesion of oral pathogens to gingival keratinocytes and (iii) the antimicrobial and antibiofilm effect of different toothpaste formulations. EEPg decreased the levels of interleukin (IL)-1β and increased IL-10 in MONO-MAC cells challenged with a periodontal biofilm. In contact with TIGK cells, EEPg reduced the numbers of adherent Porphyromonas gingivalis to 0.5% but did not affect the adhesion of Candida albicans. The frequent brushing of a cariogenic biofilm with a toothpaste supplemented with EEPg reduced the surface microhardness loss of enamel specimens. Mixing an experimental erythritol toothpaste with 25 and 50 mg/mL of EEPg confirmed the antibacterial activity of EEPg against oral bacteria and particularly inhibited periodontal biofilm formation. The suggested toothpaste formulations seem to have potential in the prevention of caries, gingivitis and periodontitis and should be evaluated in further in vitro research and in clinical trials.

Removal effect of Candida albicans biofilms from the PMMA resin surface by using a manganese oxide nanozyme-doped diatom microbubbler

To prevent oral candidiasis, removal of the Candida biofilms from dentures is important. However, common denture cleaners are insufficiently effective in removing biofilms. A manganese oxide (MnO2) nanozyme-doped diatom microbubbler (DM) can generate oxygen gas microbubbles by a catalase-mimicking activity in hydrogen peroxide (H2O2). DM can invade and destroy biofilms with the driving force of continuously generated microbubbles. In this study, the Candida biofilm removal efficiency by co-treatment of DM and H2O2 was investigated. Diatom particles were reacted with (3-aminopropyl)triethoxysilane to prepare amine-substituted diatom particles. These particles were reacted with potassium permanganate to fabricate DMs. The morphology and components of DM were analyzed by using a scanning electron microscope (SEM). Four types of denture base resin specimens on which biofilms of Candida albicans were formed were treated with phosphate-buffered saline (PBS group), Polident 5-Minute (Polident group), 0.12% chlorhexidine gluconate (CHX group), 3% H2O2 (H2O2 group), and co-treatment of 3 mg/mL of DM and 3% H2O2 (DM group). The biofilm removal effect of each group was quantitatively analyzed by crystal violet assay, and the results were visually confirmed by SEM images. After each treatment, the remaining C. albicans were stained with Hoechst 33342/propidium iodide, and observed with confocal laser scanning microscopy (CLSM) to evaluate the viability. MnO2 nanozyme sheets were successfully doped on the surface of the fabricated DM. Although biofilms were not effectively removed in the Polident and CHX groups, CLSM images showed that CHX was able to effectively kill C. albicans in the biofilms on all resin specimen types. According to the crystal violet analysis, the H2O2 groups removed the biofilms on heat-activated and 3D-printed resins (P < .01), but could not remove the biofilms on autopolymerizing and milled resins significantly (P = .1161 and P = .1401, respectively). The DM groups significantly removed C. albicans from all resin specimen types (P < .01).

High throughput bioanalytical techniques for elucidation of Candida albicans biofilm architecture and metabolome

Candida albicans is the fungus most commonly associated with biofilm formation in immunocompromised individuals, and biofilm formation is now well-recognized as a primary virulence factor in candidiasis. C. albicans biofilm production is a highly regulated and coordinated process, with adhesive contacts, morphogenetic conversions, and consortia activity all playing important roles. The cells within the biofilm are well protected from myriads of environmental stressors, such as host immune defenses and antifungal treatment, which challenges substantial clinical implications for the treatment of biofilm-associated infections. The biofilm lifestyle grounds a hotspot for more invasive and recurrent Candidal infection, as a mature biofilm releases naïve yeast cells out from the matured biofilm. For the research of biofilm and biofilm-related properties, high-throughput techniques such as spectroscopy, polymerase chain reaction (PCR), microscopy, and DNA microarray were used. Even with a small number of experimental samples, high throughput procedures provide magnified and accurate results. For their experimental samples, these biotechniques are extremely sensitive. Microscopic study of Candidal biofilms elucidates biofilm morphology, and electron microscopy can be used to produce higher resolution images of biofilms. Similarly, spectroscopic techniques aid in the identification and quantification of the primary and secondary metabolites produced by the biofilm. Genomic profiling using DNA microarray and PCR helps in analyzing the expression of various genes associated with biofilm formation. The present review comprises all the high throughput biotechniques used so far for the study of the Candidal biofilm, which will develop a sound structural understanding of the biofilm to assist in easy diagnosis and its possible treatments.

Synergistic Interaction of Caspofungin Combined with Posaconazole against FKS Wild-Type and Mutant Candida auris Planktonic Cells and Biofilms.

Candida auris is a potential multidrug-resistant pathogen able to cause biofilm-associated outbreaks, where frequently indwelling devices are the source of infections. The number of effective therapies is limited; thus, new, even-combination-based strategies are needed. Therefore, the in vitro efficacy of caspofungin with posaconazole against FKS wild-type and mutant Candida auris isolates was determined. The interactions were assessed utilizing the fractional inhibitory concentration indices (FICIs), the Bliss model, and a LIVE/DEAD assay. Planktonic minimum inhibitory concentrations (pMICs) for the caspofungin-posaconazole combination showed a 4- to 256-fold and a 2- to 512-fold decrease compared to caspofungin and posaconazole alone, respectively. Sessile minimum inhibitory concentrations (sMICs) for caspofungin and posaconazole in combination showed an 8- to 128-fold and a 4- to 512-fold decrease, respectively. The combination showed synergy, especially against biofilms (FICIs were 0.033-0.375 and 0.091-0.5, and Bliss cumulative synergy volumes were 6.96 and 32.39 for echinocandin-susceptible and -resistant isolates, respectively). The caspofungin-exposed (4 mg/L) C. auris biofilms exhibited increased cell death in the presence of posaconazole (0.03 mg/L) compared to untreated, caspofungin-exposed and posaconazole-treated biofilms. Despite the favorable effect of caspofungin with posaconazole, in vivo studies are needed to confirm the therapeutic potential of this combination in C. auris-associated infections.

Biofilm Formation on Intrauterine Devices in Diabetic and Nondiabetic Patients with Vulvovaginal Candidiasis

Abstract Introduction/Objective Women with diabetes mellitus (DM) are more susceptible to vulvovaginal candidiasis (VVC) than non-diabetics. This could be due to higher glucose levels predisposing VVC by damaging basic host defense mechanisms and stimulating Candida adhesion to vaginal cells. Objectives Investigating the prevalence of Candida albicans and biofilm formation in diabetic and nondiabetic IUD users. Methods/Case Report A total of 325 vaginal swabs and IUDs were enrolled in this study from the Delta region in Egypt. Females with a median age of 17 to 45 years were divided into two groups; diabetic (29.8%) and nondiabetic (70.1%). They were all vaginitis having one of these symptoms; irritation on the surface of the genital tract, uterine cervix ulcer, vaginal bleeding, and acute vaginal exudates. C. albicans were isolated and identified using cultural characters. Biofilm formation by Candida on removed IUDs and swabs was examined by XTT-reduction assay, scanning electron microscope (SEM), and transmission electron microscope (TEM). Antifungal susceptibility to fluconazole and amphotericin B (AMB) to isolated strains and in vitro experimental biofilm formation was performed. Results (if a Case Study enter NA) In diabetic cases, the highest percentage (91.1%) of isolated C.albicans was found among patients with irritation on the surface of the genital tract, whereas the lowest one (83.3%) was in the symptoms of acute vaginal exudates. In nondiabetic cases, the highest percentage of isolated C.albicans (51.7%) was found among patients with symptoms of acute vaginal exudates. In contrast, the lowest one (32.5%) was in the symptoms of irritation on the surface of the genital tract. XTT method illustrated the biofilm formation; SEM revealed a heterogeneous thick biofilm. Biofilm MIC to strains with AMB &amp;gt;1μg\ml was considered resistant, while those for which AMB concentration were ≤1μg\ml were deemed sensitive. Strains with Fluconazol 64 μg\ml were considered resistant, while those for which Fluconazol concentration was ≤8 μg\ml were deemed sensitive. Conclusion There was a significant direct relationship between diabetic cases and C.albicans prevalence in the current study. Furthermore, the ability to form biofilm was found to be the same in diabetic and nondiabetic patients.

In Vitro Activity of Clinically Used Farnesol and Other Antifungals Against Pre-formedCandida albicans Biofilms

Abstract Introduction/Objective Introduction: The emergence of antifungal resistance patients after long-term antifungal treatment shows an urgent need to develop new antifungal therapies. The ability of Candida albicans cells to form biofilm is resistant to many antifungal agents; it causes about 90% of vaginal yeast infections. Farnesol has a critical regulatory (quorum-sensing) molecule in C. albicans biofilm formation, a phenomenon that may be relevant to C. albicans biofilm formation. This study aims to prophesy the effect of Farnesol against pre-formed Candida albicans biofilms on the Intra-uterine devices (IUDs) users in Egypt. Methods/Case Report The study was carried out in The Delta region, Egypt, and included 325 females using IUDs with vaginitis syndromes with a median age of 20 to 45 years. About 291 (58.75%) Candida albicans were isolated from all cases. Vaginal swabs and IUDs were collected. The cultural characters identified the isolated organisms; the formation of biofilm in vitro was identified by (polystyrene multiwell microtitre plates (96 flat bottoms wells &amp; Polymetheylmethacrylate strips (PMMA)). Antifungal susceptibility testing of plaktonic cells was identified by (The broth microdilution method, Spectrophotometric Modified Microdilution Method, and Colorimetric Modified Microdilution Method). In vitro activity of clinically used fluconazole, amphotericin B, and farnesol against pre-formed biofilms formed using RPMI was assessed using the XTT-reduction assay. Results (if a Case Study enter NA) Values of Candida biofilm untreated with antifungals were 11 independent biofilms formed in each of 8 different rows of the same 96 wells microtiter plate. The experiments showed the increased resistance of sessile C. albicans cells to fluconazole and amphotericin B. Farnesol was the only antifungal agent which showed potent activity indicated by the low SMIC80 values (0.03125μg/ml). Conclusion This study highlights the drastic reduction of Franesol on C.albicans biofilm. We recommend using Franesol as an antifungal agent for C.albicans in Egypt.

Vaginal Lactobacillus Impair Candida Dimorphic Switching and Biofilm Formation.

Lactobacillus spp. generally dominate the vaginal microbiota and prevent pathogen adhesion and overgrowth, including Candida spp., by various mechanisms. Although Candida spp. can be commensal, in certain conditions they can become pathogenic, causing vulvovaginal candidiasis. The insurgence of candidiasis is related to the expression of Candida virulence factors, including morphologic switching and biofilm formation. Germ tubes, pseudohyphae, and hyphae promote Candida tissue invasion, biofilms increase persistence and are often resistant to antifungals and host immune response. Here, we explored the inhibitory activity of vaginal Lactobacillus strains belonging to Lactobacillus crispatus, Lactobacillus gasseri, Limosilactobacillus vaginalis, and Lactiplantibacillus plantarum species towards Candida virulence factors. With the aim to investigate the interrelation between mode of growth and functionality, supernatants were collected from lactobacilli planktonic cultures and, for the first time, from adherent ones, and were evaluated towards Candida dimorphic switching and biofilm. Candida biofilms were analyzed by multiple methodologies, i.e., crystal violet staining, MTT assay, and confocal microscopy. Lactobacillus supernatants reduce Candida switching and biofilm formation. Importantly, L. crispatus supernatants showed the best profile of virulence suppression, especially when grown in adherence. These results highlight the role of such species as a hallmark of vaginal eubiosis and prompt its employment in new probiotics for women’s health.

Azole-Based Compounds That Are Active against Candida Biofilm: In Vitro, In Vivo and In Silico Studies.

Fungal pathogens, including Candida spp., Aspergillus spp. and dermatophytes, cause more than a billion human infections every year. A large library of imidazole- and triazole-based compounds were in vitro screened for their antifungal activity against C. albicans, C. glabrata, C. krusei, A. fumigatus and dermatophytes, such as Microsporum gypseum, Trichophyton rubrum and Trichophyton mentagrophytes. The imidazole carbamate 12 emerged as the most active compound, showing a valuable antifungal activity against C. glabrata (MIC 1–16 μg/mL) and C. krusei (MIC 4–24 μg/mL). No activity against A. fumigatus or the dermatophytes was observed among all the tested compounds. The compound 12 inhibited the formation of C. albicans, C. glabrata and C. krusei biofilms and reduced the mature Candida biofilm. In the Galleria mellonella larvae, 12 showed a significant reduction in the Candida infection, together with a lack of toxicity at the concentration used to activate its antifungal activity. Moreover, the in silico prediction of the putative targets revealed that the concurrent presence of the imidazole core, the carbamate and the p-chlorophenyl is important for providing a strong affinity for lanosterol 14α-demethylase (CgCYP51a1) and the fungal carbonic anhydrase (CgNce103), the S-enantiomer being more productive in these interactions.

All-Trans Retinoic Acid Effect on Candida albicans Growth and Biofilm Formation.

Candida albicans (C. albicans) is the most common fungal pathogen causing recurrent mucosal and life-threatening systemic infections. The ability to switch from yeast to hyphae and produce biofilm are the key virulence determinants of this fungus. In fact, Candida biofilms on medical devices represent the major risk factor for nosocomial bloodstream infections. Novel antifungal strategies are required given the severity of systemic candidiasis, especially in immunocompromised patients, and the lack of effective anti-biofilm treatments. Retinoids have gained attention recently due to their antifungal properties. Material and methods: The present study aimed at evaluating the in vitro effects of different concentrations (300 to 18.75 µg/mL) of All-trans Retinoic Acid (ATRA), a vitamin A metabolite, on Candida growth and biofilm formation. Results: ATRA completely inhibited the fungal growth, by acting as both fungicidal (at 300 µg/mL) and fungistatic (at 150 µg/mL) agent. Furthermore, ATRA was found to negatively affect Candida biofilm formation in terms of biomass, metabolic activity and morphology, in a dose-dependent manner, and intriguingly, its efficacy was as that of amphotericin B (AmB) (2–0.12 μg/mL). Additionally, transmission electron microscopy (TEM) analysis showed that at 300 μg/mL ATRA induced plasma membrane damage in Candida cells, confirming its direct toxic effect against the fungus. Conclusion: Altogether, the results suggest that ATRA has a potential for novel antifungal strategies aimed at preventing and controlling biofilm-associated Candida infections.

Surface modification of poly(methyl-methacrylate) with farnesol to prevent Candida biofilm formation.

Candida albicans promotes biofilm formation on dentures, which compromises the use of poly(methyl-methacrylate) (PMMA) as a dental material. Farnesol (FAR), a natural compound that prevents C. albicans filamentation and biofilm formation, was incorporated into the PMMA matrix, to obtain antifungal PMMA_FAR materials. The tested concentrations (0·0125% and 0·4%) of FAR, 24 h after incubation on YPD agar, inhibited filamentation of C. albicans. PMMA was modified with different FAR concentrations (3-12%), and physicochemical properties, antifungal activity and cytotoxicity of these modified materials (PMMA_FAR) were tested. The presence of FAR in PMMA_FAR composites was verified by Fourier-transform infrared spectroscopy (FT-IR). Incorporation of FAR into the polymeric matrix significantly decreased hydrophilicity at all tested concentrations and significantly reduced biofilm and planktonic cells metabolic activity in the early stage of biofilm formation at ≥6% FAR in PMMA. PMMA_FAR composites with <9% FAR were non-toxic. Modification of PMMA with FAR is a good strategy for reducing C. albicans biofilm formation on dentures.

P060 A preliminary in vitro and in vivo evaluation of the effect and action mechanism of 17-AAG combined with azoles against azole-resistant Candida spp.

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMInvasive candidiasis is the primary reason for the increasing cases of mortality in a medical environment. The resistance spectra of Candida species to antifungal drugs, among which Candida auris is the most prominent, have gradually expanded.ObjectivesHsp90 plays a protective role in the stress response of fungi and facilitates their virulence. In contrast, Hsp90 inhibitors can improve the resistance of fungi to antifungal drugs by regulating the heat resistance of Hsp90 and thereby destroying the integrity of the fungal cell walls. Therefore, we used Hsp90 inhibitor in combination with different antifungal drugs to explore its antifungal effect and mechanism.MethodsThe drugs tested for the resistance included itraconazole, voriconazole, posaconazole, fluconazole, and 17-AAG. A total of 20 clinical strains of Candida were investigated. The broth microdilution checkerboard technique, as adapted from the CLSI M27-A4 method, was applied in this study. At the same time, the effect of 17-AAG combined with antifungal drugs on the formation of Candida biofilm was observed, and the animal experiment of C. mellonella was carried out in vivo. Moreover, we determined that with the use of rhodamine 6 G to detect drug efflux and that of dihydrorhodamine-123 to detect intracellular reactive oxygen species (ROS).ResultsWe found that 17-AAG alone exerted limited antifungal activity against all tested strains. The MIC range of 17-AAG was 8 to >32 μg/ml. The synergy among 17-AAG and itraconazole, voriconazole, and posaconazole was observed against 10 (50%), 7 (35%), and 13 (65%) of all isolates, respectively. Moreover, the synergy between 17-AAG and fluconazole was observed against 5 (50%) stains of azole-resistant Candida. However, no antagonism was recorded. In vivo test, the combination group also significantly prolonged the infection event and improved the survival of larvae. Treatment with 17-AAG combined with azole drugs inhibited the efflux pump of fungi and promoted the accumulation of ROS in the fungal cells.ConclusionOur result adequately verifies the influence of 17-AAG on the formation of Candida spp. biofilm. The mechanism of 17-AAG combined with azoles could kill fungi by inhibiting drug efflux and increasing intracellular reactive oxygen species. These results thereby provide a new idea to further explore drugs against drug-resistant Candida spp.

P068 Inhibition of Candida glabrata biofilm using the combination of a novel antifungal cyclic lipopeptide and azole: An in vitro synergistic approach

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMThe general feature of the biofilm is a densely packed microbial community with an extracellular matrix composed of polysaccharides and proteins. Candida glabrata was reported as the most biofilm-forming species responsible for causing virulent Candida infections among Candida non-albicans (CNA). Candida glabrata is the most azole-resistant species of all Candida species. The early and maturing biofilms enable the Candida cells to overcome the effects of azoles and exhibit higher drug resistance. Since simple azole mono-therapy rarely eradicates recalcitrant Candida biofilms, removal of the infected device becomes necessary for curing the infection.ObjectiveThe objective was to assess the biofilm inhibition effect of antifungal cyclic lipopeptide which has been purified by a multi-step process from the cell-free supernatant of Bacillus subtilis, and to analyze the synergistic activity of the purified lipopeptide lead compound designated as AF4 with the standard antifungal fluconazole using different concentrations.MethodsBriefly, cell-free supernatant was extracted using a solvent mixture and silica gel-based adsorption chromatography. The potential antifungal cyclic lipopeptide variant AF4 was extracted from the soil isolate B. subtilis by a multi-step purification process that involves reversed-phase HPLC and exhibited a wide-spectrum of anti-Candida activity. The XTT reduction assay was used for metabolically active cells after treatment with a combination of drugs, and crystal violet (CV) assay was performed to quantify the cell biomass. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to analyze the biofilm morphology and architecture of the AF4 lipopeptide at (2xMIC) and fluconazole (8x-16xMIC)-treated biofilms. For confocal microscopy-based biofilm visualization, a combination of two different dyes was used. Extracellular polymeric substances were stained by concanavalin A-Alexa Fluor 488 conjugate and, the FUN- 1 fluorescent dye was used for staining the live biofilm-forming Candida cells. After capturing the Z-stack images in CLSM, the biomass (μm3/μm2), average thickness (μm), and roughness of the biofilm were analyzed by Comstat 2.1 software.ResultsThe AFST analysis showed that the MIC of the lead antifungal lipopeptide was 8 μg/mL against the C. glabrata planktonic cells. Adding the antifungal compound at higher MICs such as by 2-folds exhibited strong antibiofilm effect that significantly enhanced the synergistic antifungal activity of the fluconazole against C. glabrata biofilm formation. Synergistic effect of fluconazole at 8x-16xMIC with 2xMIC of the lead compound AF4 on C. glabrata 24 h maturing biofilm had increased significantly as revealed by the XTT and CV assays. The results confirmed ˃ 50% inhibition of the biofilm in both 6 h developmental and 24 h maturing biofilms in 8xMIC fluconazole plus 2xMIC AF4 while compared with fluconazole alone at the same concentration. The results from the XTT and, CV assays and SEM and CLSM (Fig. 1) suggest that the combinatorial treatments of the lead antifungal lipopeptide and fluconazole at varying concentrations have the potential of eradicating or nearly eradicating the maturing biofilm of CAN.ConclusionsThe combinatorial study was found effective in combating the biofilm formation. A correlation between metabolic activity and antifungal resistance in maturing biofilms has been found.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on &lt;em&gt;Candida tropicalis&lt;/em&gt; Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less. It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm. The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Combining Essential Oils with Each Other and with Clotrimazole Prevents the Formation of Candida Biofilms and Eradicates Mature Biofilms.

Fungal infections by Candida spp. are opportunistic and most often occur in individuals with some predisposing factor. Essential oils (EO) have anti-Candida potential, being a therapeutic alternative to be explored, especially for superficial and mucosal candidiasis. The objective was to analyze the synergistic potential between the EO of Citrus limon, Cupressus sempervirens, Litsea cubeba and Melaleuca alternifolia, and each of them with clotrimazole, to inhibit in vitro the formation and eradication of Candida spp. biofilms. Added to this, the survival of Caenorhabditis elegans was evaluated after exposure to EO, clotrimazole and their synergistic combinations. Anti-Candida activity was determined by microdilution for the substances alone and in EO–EO and EO–clotrimazole combinations. The combinations were performed by the checkerboard method, and the reduction in the metabolic activity of biofilms was determined by the viability of MTT/menadione. C. elegans larvae survival was evaluated after 24 h of exposure to EO, clotrimazole and synergistic combinations. The minimum inhibitory concentration (MIC) of EO ranged from 500 to >4000 µg/mL. The lowest MIC (500 µg/mL) was for C. sempervirens and L. cubeba on a C. krusei isolate; for clotrimazole, the MIC ranged from 0.015 to 0.5 µg/mL. Biofilm inhibition and eradication both ranged from 1000 to >4000 µg/mL. The lethal concentration (LC50) of C. limon, L. cubeba and M. alternifolia was 2000 µg/mL for C. elegans, while for C. sempervirens and clotrimazole, it was not determined within the concentration limits tested. In combination, more than 85% of the larvae survived M. alternifolia–clotrimazole, M. alternifolia–L. cubeba, C. sempervirens–clotrimazole and C. sempervirens–C. limon combinations. This study is the first, to our knowledge, to present a synergistic relationship of EO–EO and EO–clotrimazole combinations on Candida spp. biofilms.

Silver-loaded mesoporous silica nanoparticles enhanced the mechanical and antimicrobial properties of 3D printed denture base resin

The aim of this study is to develop a novel 3D printed denture base resin material modified with mesoporous silica nanocarrier loaded with silver (Ag/MSN) to enhance mechanical and antimicrobial properties. Acrylate resin-based was incorporated with various proportion of Ag/MSN (0.0–2.0 wt%). Specimens with different geometry were printed and characterized accordingly for the effect of modification on properties such as: mechanical and physical properties, chemical composition and degree of conversion, as well as biological response in term of biocompatibility and antimicrobial against oral fibroblast and candida biofilm (C. albicans), respectively. The consecutive addition of Ag/MSN improved significantly surface hardness and crack propagation resistance, while flexural strength remained similar to control; however, a negligible decrease was observed with higher concentrations ≥1 wt%. No significant difference was noticed with water sorption, while water solubility had a remarkable trend of reduction associated with filler content. The surface roughness significantly increased when concentration of Ag/MSN was ≥1.0 wt%. A significant reduction in C. albicans biofilm mass, as the inhibition proficiency was correlated with the proportion of the filler. With respect to the amount of Ag/MSN, the modification was compatible toward fibroblast cells. The sequential addition of Ag/MSN enhanced significantly the mechanical and antimicrobial properties of the 3D printed resin-based material without affecting adversely compatibility. The acrylic resin denture base material has susceptibility of microbial adhesion which limits its application. Silver loaded MSN showed a significant performance to enhance antimicrobial activity against C. albicans which is the main cause of denture stomatitis. The proposed invention is a promise technique for clinical application to provide an advanced prosthesis fabrication and serve as long-term drug delivery.

In vivo antifungal activity and biocompatibility of Cryptocarya moschata.

The objective of this study was evaluate, in vivo model, the antifungal activity of Cryptocarya moschata extract against Candida albicans and its biocompatibility. The animals (N = 50) were divided into groups (n = 5): CI/CG: candidiasis was induced and treated with C. moschata extract (0.045g/mL); CI/NG: candidiasis was induced and treated with nystatin; CI/NT: candidiasis was induced and no treated; CI/CG-2: candidiasis was induced and treated with C. moschata extract (0.045g/mL), reapplied after 24h; CI/NG-2: candidiasis was induced and treated with nystatin, reapplied after 24h; NCI/NT: candidiasis was not induced and no treated; NCI/CG: candidiasis was not induced and treated with C. moschata extract (0.045g/mL); NCI/NG: candidiasis was not induced treated with nystatin; NCI/CG-2: candidiasis was not induced and treated with C. moschata extract (0.045g/mL), reapplied after 24h; NCI/NG-2: candidiasis was not induced and treated with nystatin, reapplied after 24h. The fungi present in the lingual dorsum of mice were collected and analyzed by the count of colony-forming units. In addition, histological analysis was performed. Histologically, there was no cell damage in the mice's tongue, and there was a decrease in Candida biofilm, similar to the use of nystatin. It was concluded that the C. moschata extract was effective against C. albicans and was biocompatible.

Niclosamide-loaded nanoparticles disrupt Candida biofilms and protect mice from mucosal candidiasis.

Candida albicans biofilms are a complex multilayer community of cells that are resistant to almost all classes of antifungal drugs. The bottommost layers of biofilms experience nutrient limitation where C. albicans cells are required to respire. We previously reported that a protein Ndu1 is essential for Candida mitochondrial respiration; loss of NDU1 causes inability of C. albicans to grow on alternative carbon sources and triggers early biofilm detachment. Here, we screened a repurposed library of FDA-approved small molecule inhibitors to identify those that prevent NDU1-associated functions. We identified an antihelminthic drug, Niclosamide (NCL), which not only prevented growth on acetate, C. albicans hyphenation and early biofilm growth, but also completely disengaged fully grown biofilms of drug-resistant C. albicans and Candida auris from their growth surface. To overcome the suboptimal solubility and permeability of NCL that is well known to affect its in vivo efficacy, we developed NCL-encapsulated Eudragit EPO (an FDA-approved polymer) nanoparticles (NCL-EPO-NPs) with high niclosamide loading, which also provided long-term stability. The developed NCL-EPO-NPs completely penetrated mature biofilms and attained anti-biofilm activity at low microgram concentrations. NCL-EPO-NPs induced ROS activity in C. albicans and drastically reduced oxygen consumption rate in the fungus, similar to that seen in an NDU1 mutant. NCL-EPO-NPs also significantly abrogated mucocutaneous candidiasis by fluconazole-resistant strains of C. albicans, in mice models of oropharyngeal and vulvovaginal candidiasis. To our knowledge, this is the first study that targets biofilm detachment as a target to get rid of drug-resistant Candida biofilms and uses NPs of an FDA-approved nontoxic drug to improve biofilm penetrability and microbial killing.

Production of new antimicrobial palm oil-derived sophorolipids by the yeast Starmerella riodocensis sp. nov. against Candida albicans hyphal and biofilm formation

BackgroundMicrobial derived-surfactants display low eco-toxicity, diverse functionality, high biodegradability, high specificity, and stability under extreme conditions. Sophorolipids are emerging as key biosurfactants of yeast origins, used in various industrial sectors to lower surface tension. Recently, sophorolipid complexes have been applied in biomedicals and agriculture to eradicate infectious problems related to human and plant fungal pathogens. This study aimed to characterize the functional properties and antifungal activities of sophorolipids produced by a newly characterized Starmerella riodocensis GT-SL1R sp. nov. strain.ResultsStarmerella riodocensis GT-SL1R sp. nov. strain was belonged to Starmerella clade with 93.12% sequence similarity using the ITS technique for strain identification. Sophorolipids production was examined, using co-carbon substrates glucose and palm oil, with a yield on the substrate between 30 and 46%. Using shake-flasks, the S. riodocensis GT-SL1R strain produced biosurfactants with an emulsification activity of 54.59% against kerosene compared to the S. bombicola BCC5426 strain with an activity of 60.22%. Maximum productivities of GT-SL1R and the major sophorolipid-producer S. bombicola were similar at 0.8 gl−1 h−1. S. riodocensis GT-SL1R produced mixed forms of lactonic and acidic sophorolipids, shown by TCL, FTIR, and HPLC. Importantly, the complex sophorolipid mixture displayed antifungal activity against an opportunistic yeast pathogen Candida albicans by effectively reducing hyphal and biofilm formation.ConclusionsSophorolipids derived from S. riodocensis demonstrate potential industrial and biomedical applications as green surfactant and antifungal agent. Since numerous renewable bioresources and industrial wastes could be used by microbial cell factories in the biosynthesis of biosurfactants to reduce the production cost, sophorolipids hold a promising alternative to current antimicrobials in treatments against infectious diseases in humans, animals, and plants.

Eugenol: A novel therapeutic agent for the inhibition of Candida species infection.

The high occurrence and mortality rates related to candidiasis emphasize the urgent need to introduce new therapeutic approaches to treat this infection. Eugenol, the main phenolic component of Clove and Cinnamomum essential oil, has been used to inhibit growth and different virulence factors of Candida, including strains with decreased susceptibility to antifungals, particularly fluconazole. The results showed that this compound could bind to Candida membrane and decrease ergosterol biosynthesis, consequently leading to cell wall and membrane damage. Additionally, eugenol not only reduced germ tube formation, which reduces nutrient absorption from host tissues, but it also increased the levels of lipid peroxidation and reactive oxygen species, which induces oxidative stress and causes high permeability in the fungal cell membrane. Eugenol inhibited Candida cells’ adhesion capacity; additionally, this compound inhibited the formation of biofilms and eliminated established Candida biofilms on a variety of surfaces. Furthermore, by disrupting fungal cell integrity, eugenol could boost the entry of the antifungal drugs into the Candida cell, improving treatment efficacy. Therefore, eugenol could be used in the clinical management of various presentations of candidiasis, especially mucocutaneous presentations such as oral and vulvovaginal infections. However, further investigations, including in vivo and animal studies, toxicology studies and clinical trials, as well as molecular analysis, are needed to improve formulations and develop novel antifungal agents based on eugenol.