Candida Albicans Biofilm Development Research Articles

Chemical composition, antifungal and antibiofilm activities of essential oils from Glycyrrhiza foetida (Desf.) growing in Tunisia.

This study was designated to investigate the chemical composition, the antifungal activity and antibiofilm properties of Glycyrrhiza foetida (Desf.) growing in Tunisia and recognized for its pharmacological and therapeutic effects. The chemical analysis of essential oil samples prepared via hydrodistillation of the aerial parts was performed by gas chromatography-mass spectrometry (GC-MS). Moreover, the antifungal activity of G. foetida essential oil was developed against three dermatophyte strains, two molds and Candida spp. yeasts using the broth microdilution assay. According to the percentages, the main constituents are δ-cadinene (13.9%), (E)-caryophyllene (13.2%) and γ-cadinene (8.3%). The efficiency of the essential oil in inhibiting Candida albicans biofilms formation was also evaluated in terms of inhibitory percentages. The results showed that C. albicans and Microsporum canis were the most sensitive to G. foetida essential oil with a complete inhibition at 0.4 and 0.2mg ml-1 , respectively. Candida albicans biofilm development was reduced by 80% by the volatile oil at a concentration of 0.8mg ml-1 . The essential oil of G. foetida has a promising role in the control of fungal agents with medical interest and in inhibition of Candida biofilm development.

Candida albicans biofilm development is governed by cooperative attachment and adhesion maintenance proteins

The opportunistic fungal pathogen Candida albicans is capable of adhering to the oral mucosa despite forces created by salivary flow. Although many fungal adhesion proteins have been identified, less is known about the temporal development of cell adhesion and biofilm growth in a flow environment. In this study, we use a flow system with real-time imaging of C. albicans cells as they adhere and grow. Rates of cell attachment and dispersion of C. albicans knockout strains of putative adhesins, transcription factors, and deletions with a hyperfilamentous phenotype were quantified during 18 h of biofilm development. Cell adhesion under flow is a multi-phase process initiated with cell rolling, then an initial firm attachment to the substrate occurs. After attachment, cells enter a growth phase where cells either commit to adherence or disperse. C. albicans Δeap1, Δhwp2, Δhyr1, and Δihd1 cells had significantly reduced initial attachment and subsequent adhesion, while Δals1/Δals3 had no change in initial attachment but reduced adhesion maintenance. WT cells had increased adhesion during the late growth phase when hyphae were more highly expressed. Hyperfilamentous strains had 10-fold higher total biofilm growth, a result of significantly reduced detachment rates, showing that hyphal morphogenesis is important for adhesion maintenance in the developing biofilm. The rate of C. albicans biomass dispersion was most important for determining the density of the mature biomass. Adhesion maintenance was mediated in part by Ywp1, a protein previously thought to regulate dispersion, thus it functions as an adhesion maintenance protein in C. albicans.

Effects of the binding of a Helianthus annuus lectin to Candida albicans cell wall on biofilm development and adhesion to host cells

BackgroundIn our previous study, we isolated and characterized a lectin called Helja from Helianthus annuus (sunflower) and then, in a further study, demonstrated its antifungal activity against Candida spp. Since Candida infections are a major health concern due to the increasing emergence of antifungal resistant strains, the search for new antifungal agents offers a promising opportunity for improving the treatment strategies against candidiasis. PurposeThe aim of this work was to get insights about the mechanism of action of Helja, an antifungal lectin of H. annuus, and to explore its ability to inhibit Candida albicans biofilm development and adherence to buccal epithelial cells (BEC). Study design/methodsYeast viability was evaluated by Evans Blue uptake and counting of colony forming units (CFU). The yeast cell integrity was assessed using Calcofluor White (CFW) as a cell wall perturbing agent and sorbitol as osmotic protectant. The induction of oxidative stress was evaluated using 3,3′-diaminobenzidine (DAB) for detection of hydrogen peroxide. The adherence was determined by counting the yeast cells attached to BEC after methylene blue staining. The biofilms were developed on polystyrene microplates, visualized by confocal laser scanning microscopy and the viable biomass was quantified by CFU counting. The binding lectin-Candida was assessed using Helja conjugated to fluorescein isothiocyanate (Helja-FITC) and simultaneous staining with CFW. The cellular surface hydrophobicity (CSH) was determined using a microbial adhesion to hydrocarbons method. ResultsC. albicans cells treated with 0.1 µg/µl of Helja showed a drastic decrease in yeast survival. The lectin affected the fungal cell integrity, induced the production of hydrogen peroxide and inhibited the morphological transition from yeast to filamentous forms. Helja caused a significant reduction of adherent cells and a decrease in biofilm biomass and coverage area. The treatment with the protein also reduced the surface hydrophobicity of fungal cells. We show the binding of Helja-FITC to yeast cells distributed as a thin outer layer to the CFW signal, and this interaction was displaced by mannose and Concanavalin A. ConclusionThe results demonstrate the interaction of Helja with the mannoproteins of C. albicans cell wall, the disruption of the cell integrity, the induction of oxidative stress, the inhibition of the morphological transition from yeast to filamentous forms and the fungal cell viability loss. The binding Helja-Candida also provides a possible explanation of the lectin effect on cell adherence, biofilm development and CSH, relevant features related to virulence of the pathogen.

Development of Candida albicans Biofilms Is Diminished by Paeonia lactiflora via Obstruction of Cell Adhesion and Cell Lysis.

Candida albicans infections are often problematic to treat owing to antifungal resistance, as such infections are mostly associated with biofilms. The ability of C. albicans to switch from a budding yeast to filamentous hyphae and to adhere to host cells or various surfaces supports biofilm formation. Previously, the ethanol extract from Paeonia lactiflora was reported to inhibit cell wall synthesis and cause depolarization and permeabilization of the cell membrane in C. albicans. In this study, the P. lactiflora extract was found to significantly reduce the initial stage of C. albicans biofilms from 12 clinical isolates by 38.4%. Thus, to assess the action mechanism, the effect of the P. lactiflora extract on the adhesion of C. albicans cells to polystyrene and germ tube formation was investigated using a microscopic analysis. The density of the adherent cells was diminished following incubation with the P. lactiflora extract in an acidic medium. Additionally, the P. lactiflora-treated C. albicans cells were mostly composed of less virulent pseudohyphae, and ruptured debris was found in the serum-containing medium. A quantitative real-time PCR analysis indicated that P. lactiflora downregulated the expression of C. albicans hypha-specific genes: ALS3 by 65% (p = 0.004), ECE1 by 34.9% (p = 0.001), HWP1 by 29.2% (p = 0.002), and SAP1 by 37.5% (p = 0.001), matching the microscopic analysis of the P. lactiflora action on biofilm formation. Therefore, the current findings demonstrate that the P. lactiflora ethanol extract is effective in inhibiting C. albicans biofilms in vitro, suggesting its therapeutic potential for the treatment of biofilm-associated infections.

The Anti-Adhesive Effect of Curcumin on Candida albicans Biofilms on Denture Materials

The use of natural compounds as an alternative source of antimicrobials has become a necessity given the growing concern over global antimicrobial resistance. Polyphenols, found in various edible plants, offers one potential solution to this. We aimed to investigate the possibility of using curcumin within the context of oral health as a way of inhibiting and preventing the harmful development of Candida albicans biofilms. We undertook a series of adsorption experiments with varying concentrations of curcumin, showing that 50 μg/ml could prevent adhesion. This effect could be further synergized by the curcumin pre-treatment of yeast cells to obtain significantly greater inhibition (>90%, p < 0.001). Investigation of the biological impact of curcumin showed that it preferentially affected immature morphological forms (yeast and germlings), and actively promoted aggregation of the cells. Transcriptional analyses showed that key adhesins were down-regulated (ALS1 and ALS3), whereas aggregation related genes (ALS5 and AAF1) were up-regulated. Collectively, these data demonstrated that curcumin elicits anti-adhesive effects and that induces transcription of genes integrally involved in the processes related to biofilm formation. Curcumin and associated polyphenols therefore have the capacity to be developed for use in oral healthcare to augment existing preventative strategies for candidal biofilms on the denture surface.

Inhibition of Candida albicans biofilm development by unencapsulated Enterococcus faecalis cps2

Background/purposeIn the oral environment, Candida albicans interacts with many bacteria, including Enterococcus faecalis. We investigated the susceptibility of C. albicans biofilm development to the presence of unencapsulated E. faecalis cps2 in comparison with reference strains (E. faecalis ATCC 29212) or their respective spent medium (collected at 6 hours). Material and methodsCrystal violet stain was used to measure the total biofilm mass, whereas quantitative real-time polymerase chain reaction was used to analyze the change in expression of the mRNA of hypha morphology (ALS1 and ALS3) and biofilm maturation (EFB1). ResultsAt the intermediate stage, C. albicans resisted the presence of each E. faecalis strain tested and their spent medium. However, at the maturation stage, the unencapsulated strain was stronger in reducing C. albicans biofilms than the reference strain (P < 0.05). At this maturation stage, the transcription levels of each gene tested decreased in the presence of either E. faecalis strains or their respective spent medium. The unencapsulated strain was more pronounced in reducing ALS1/ALS3 expression, whereas the respective spent medium had a similar capability to restrict the expression of EFB1. ConclusionThis study showed, the unencapsulated strain is more effective in inhibiting C. albicans biofilm development compared with the reference strains. In contrast, the secreted molecules produced by each strain tested are necessary in controlling the growths of C. albicans biofilm.

Probiotic lactobacilli inhibit early stages of Candida albicans biofilm development by reducing their growth, cell adhesion, and filamentation.

We evaluated the inhibitory effects of the probiotic Lactobacillus species on different phases of Candida albicans biofilm development. Quantification of biofilm growth and ultrastructural analyses were performed on C. albicans biofilms treated with Lactobacillus rhamnosus, Lactobacillus casei, and Lactobacillus acidophilus planktonic cell suspensions as well as their supernatants. Planktonic lactobacilli induced a significant reduction (p<0.05) in the number of biofilm cells (25.5-61.8%) depending on the probiotic strain and the biofilm phase. L. rhamnosus supernatants had no significant effect on the mature biofilm (p>0.05), but significantly reduced the early stages of Candida biofilm formation (p<0.01). Microscopic analyses revealed that L. rhamnosus suspensions reduced Candida hyphal differentiation, leading to a predominance of budding growth. All lactobacilli negatively impacted C. albicans yeast-to-hyphae differentiation and biofilm formation. The inhibitory effects of the probiotic Lactobacillus on C. albicans entailed both cell-cell interactions and secretion of exometabolites that may impact on pathogenic attributes associated with C. albicans colonization on host surfaces and yeast filamentation. This study clarifies, for the first time, the mechanics of how Lactobacillus species may antagonize C. albicans host colonization. Our data elucidate the inhibitory mechanisms that define the probiotic candicidal activity of lactobacilli, thus supporting their utility as an adjunctive therapeutic mode against mucosal candidal infections.

Modulation of Candida albicans Biofilm by Different Carbon Sources.

In the present investigation, the role of carbon sources (glucose, lactate, sucrose, and arabinose) on Candida albicans biofilm development and virulence factors was studied on polystyrene microtiter plates. Besides this, structural changes in cell wall component β-glucan in presence of different carbon sources have also been highlighted. Biofilm formation was analyzed by XTT (2,3-bis[2-Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay. Glucose-grown cells exhibited the highest metabolic activity during adhesion among all carbon sources tested (p<0.05). However, cells exposed to sucrose exhibited highest biofilm formation and matrix polysaccharides secretion after 48h. The results also correlated with the biofilm height and roughness measurements by atomic force microscopy. Exposure to lactate induced hyphal structures with the highest proteinase activity while arabinose-grown cells formed pseudohyphal structures possessing the highest phospholipase activity. Structural changes in β-glucan characterized by Fourier transform infrared (FTIR) spectroscopy displayed characteristic band of β-glucan at 892cm(-1) in all carbon sources tested. The β(1→6) to β(1→3) glucan ratio calculated as per the band area of the peak was less in lactate (1.15) as compared to glucose (1.73), sucrose (1.62), and arabinose (2.85). These results signify that carbon sources influence C. albicans biofilm development and modulate virulence factors and structural organization of cell wall component β-glucan.

Candida albicans biofilm development on medically-relevant foreign bodies in a mouse subcutaneous model followed by bioluminescence imaging.

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Influence of surface free energy of denture base and liner materials onCandida albicansbiofilms

This study aimed to evaluate the influence of surface free energy (SFE) of denture base and liner materials on Candida albicans biofilm development. Discs were fabricated using poly(methyl methacrylate) acrylic resin and poly(ethyl methacrylate) denture liner, according to the manufacturers' instructions. For SFE test, discs were pellicle-coated with saliva alone, saliva+blood plasma, or blood plasma alone. Candida albicans biofilms were allowed to form on pellicle-coated discs for 48h. Biofilms were evaluated for cell counts, metabolic activity, and structural characteristics at adhesion phase (after 1.5h of development) and at biofilm maturity (after 48h of development). Data were analyzed by anova and Tukey tests using a significance level of 5%. Saliva+blood plasma pellicles had a higher SFE compared to pellicles of saliva or blood plasma alone (P<0.001). Differences in SFE by pellicle-coating did not affect the cell counts, metabolic activity, or structure at the adhesion phase (P>0.05). In contrast, the presence of blood plasma resulted in higher cell counts, biovolume, and thickness of mature biofilms on both materials (P<0.001). Increases in SFE from pellicle-coating leads to robust mature C.albicans biofilms on both denture materials.

Influence of substratum position and acquired pellicle on Candida albicans biofilm

The purpose of this study was to evaluate the influence of the substratum position and the saliva acquired pellicle (AP) on Candida albicans biofilm development. Poly(methylmethacrylate) (PMMA) disks were fabricated and randomly allocated to experimental groups: HNP (disks placed in a horizontal position and uncoated by pellicle), VNP (disks placed in a vertical position and uncoated by pellicle), HCP (disks placed in a horizontal position and coated by pellicle), and VCP (disks placed in a vertical position and coated by pellicle). Disks were placed in a 24-well plate and a suspension of 107 cells/mL of Candida albicans was added to each well for biofilm development. The plates were aerobically incubated at 35°C. The biofilms were evaluated at 1.5 (adhesion time point), 24, 48, 72, and 96 hours. The number of viable cells was quantified in terms of the colony-forming units per milliliter (CFU/mL). Metabolic activity was measured by the XTT assay. The biofilm structure was analyzed by scanning electron microscopy. The data were analyzed by three-way ANOVA followed by Tukey's test, with significance set at 5%. The vertical groups showed less biofilm formation and lower metabolic activity than the horizontal groups (p<0.05). Significant differences in cell viability and metabolic activity were observed between the adhesion and other time points (p<0.05), but these variables were not affected by the presence of the pellicle (p>0.05). It can be concluded that the substratum position influenced biofilm development.

Involvement of Heat Shock Proteins in Candida albicans Biofilm Formation

Biofilm growth represents one of the most challenging problems associated with Candida infections, largely due to the natural resistance of biofilm to the common antifungal drugs. As elevated expression of heat shock proteins (HSP) promotes Candida yeast-hyphae switch, which is an essential step in biofilm formation, we investigated the expression of hsp genes during Candida albicans biofilm development. By measuring mRNA levels using qRT-PCR, we found that all three hsp genes that we monitored are overexpressed in the initial stage of C. albicans biofilm formation. To corroborate this finding, we examined the effect of 17-DMAG, a specific Hsp90 inhibitor, on the formation of C. albicans biofilm. Our results indicate the requirement of HSP during the early phase of Candida biofilm development.

Genetic control of Candida albicans biofilm development

Candida species cause frequent infections owing to their ability to form biofilms - surface-associated microbial communities - primarily on implanted medical devices. Increasingly, mechanistic studies have identified the gene products that participate directly in the development of Candida albicans biofilms, as well as the regulatory circuitry and networks that control their expression and activity. These studies have uncovered new mechanisms and signals that govern C. albicans biofilm development and associated drug resistance, thus providing biological insight and therapeutic foresight.

Candida albicans biofilm on titanium: effect of peroxidase precoating.

The present study aimed to document Candida albicans biofilm development on titanium and its modulation by a peroxidase-precoated material which can generate antimicrobials, such as hypoiodite or hypothiocyanite, from hydrogen peroxide, iodide, or thiocyanate. For this purpose, titanium (powder or foil) was suspended in Sabouraud liquid medium inoculated with C. albicans ATCC10231. After continuous stirring for 2-21 days at room temperature, the supernatant was monitored by turbidimetry at 600 nm and titanium washed three times in sterile Sabouraud broth. Using the tetrazolium salt MTT-formazan assay, the titanium-adherent fungal biomass was measured as 7.50 ± 0.60 × 10(6) blastoconidia per gram of titanium powder (n = 30) and 0.50 ± 0.04 × 10(6) blastoconidia per cm(2) of titanium foil (n = 12). The presence of yeast on the surface of titanium was confirmed by microscopy both on fresh preparations and after calcofluor white staining. However, in the presence of peroxidase systems (lactoperoxidase with substrates such as hydrogen peroxide donor, iodide, or thiocyanate), Candida growth in both planktonic and attached phases appeared to be inhibited. Moreover, this study demonstrates the possible partition of peroxidase systems between titanium material (peroxidase-precoated) and liquid environment (containing peroxidase substrates) to limit C. albicans biofilm formation.

Candida Biofilms: Is Adhesion Sexy?

The development of Candida albicans biofilms requires two types of adhesion molecule - the Als proteins and Hwp1. Mutational analyses have recently revealed that these molecules play complementary roles, and their characteristics suggest that they may have evolved from primitive mating agglutinins.

Temporal analysis of Candida albicans gene expression during biofilm development

Microarrays were used to identify changes in gene expression associated with Candida albicans biofilm development. Two biofilm substrates (denture and catheter), and two C. albicans strains for each substrate, were tested to remove model- and strain-dependent variability from the overall dataset. Three biofilm developmental phases were examined: early (6 h), intermediate (12 h), and mature (48 h). Planktonic specimens were collected at the same time points. Data analysis focused primarily on gene expression changes over the time-course of biofilm development. Glycolytic and non-glycolytic carbohydrate assimilation, amino acid metabolism, and intracellular transport mechanisms were important during the early phase of biofilm formation. These early events increase intracellular pools of pyruvate, pentoses and amino acids, which prepare the biofilm for the large biomass increase that begins around 12 h of development. This developmental stage demands energy and utilizes specific transporters for amino acids, sugars, ions, oligopeptides and lactate/pyruvate. At mature phase (48 h), few genes were differentially expressed compared with the 12 h time point, suggesting a relative lack of initiation of new metabolic activity. Data analysis to assess biofilm model-specific gene expression showed more dynamic changes in the denture model than in the catheter model. Data analysis to identify gene expression changes that are associated with each strain/substrate combination identified the same types of genes that were identified in the analysis of the entire dataset. Collectively, these data suggest that genes belonging to different, but interconnected, functional categories regulate the morphology and phenotype of C. albicans biofilm.

Development and characterization of an in vivo central venous catheter Candida albicans biofilm model.

Biofilms represent a niche for microorganisms where they are protected from both the host immune system and antimicrobial therapies. Biofilm growth serves as an increasing source of clinical infections. Candida infections are difficult to manage due to their persistent nature and associated drug resistance. Observations made in biofilm research have generally been limited to in vitro models. Using a rat central venous catheter model, we characterized in vivo Candida albicans biofilm development. Time-course quantitative culture demonstrated a progressive increase in the burden of viable cells for the first 24 h of development. Fluorescence and scanning electron microscopy revealed a bilayered architecture. Adjacent to the catheter surface, yeast cells were densely embedded in an extracellular matrix. The layer adjacent to the catheter lumen was less dense. The outermost surface of the biofilm contained both yeast and hyphal forms, and the extracellular material in which they were embedded appeared fibrous. These architectural features were similar in many respects to those described for in vitro models. However, scanning electron microscopy also revealed host cells embedded within the biofilm matrix. Drug susceptibility was determined by using two assays and demonstrated a biofilm-associated drug resistance phenotype. The first assay demonstrated continued growth of cells in the presence of supra-MIC antifungal drug concentrations. The second assay demonstrated reduced susceptibility of biofilm-grown cells following removal from the biofilm structure. Lastly, the model provided sufficient nucleic material for study of differential gene expression associated with in vivo biofilm growth. Two fluconazole efflux pumps, CDR1 and CDR2, were upregulated in the in vivo biofilm-associated cells. Most importantly, the studies described provide a model for further investigation into the molecular mechanisms of C. albicans biofilm biology and drug resistance. In addition, the model provides a means to study novel drug therapies and device technologies targeted to the control of biofilm-associated infections.

Role of dimorphism in the development of Candida albicans biofilms.

Two model biofilm systems, involving growth on disks of catheter material or on cylindrical cellulose filters, were used to investigate the structure of Candida albicans biofilms. To assess the importance of dimorphism in biofilm development, biofilms produced by two wild-type strains were compared with those formed by two morphological mutants, incapable of yeast and hyphal growth, respectively. Scanning electron microscopy and thin sections of biofilms examined by light microscopy revealed that biofilms of the wild-type strains formed on catheter disks consisted of two distinct layers: a thin, basal yeast layer and a thicker, but more open, hyphal layer. The hypha- mutant produced only the basal layer, whereas the yeast- mutant formed a thicker, hyphal biofilm equivalent to the outer zone of the wild-type structures. Biofilms of the yeast- mutant were more easily detached from the catheter surface than the others, suggesting that the basal yeast layer has an important role in anchoring the biofilm to the surface. Biofilms formed on cylindrical cellulose filters were quite different in appearance. The hypha- mutant and both wild types produced exclusively yeast-form biofilms whereas the yeast- mutant generated a dense hyphal mat on the top of the filter. All these biofilms, irrespective of morphological form, were resistant to the antifungal agent, amphotericin B. Overall, these results indicate that the structure of a C. albicans biofilm depends on the nature of the contact surface, but that some surfaces produce biofilms with a layered architecture resembling to that described for bacterial systems.