Eap1p, an Adhesin That Mediates Candida albicans Biofilm Formation In Vitro and In Vivo

<i>Candida albicans</i> biofilm formation and its clinical consequences

The objective of this study was to evaluate the effect of denture base resin containing silver nanoparticles (nano-silver) on Candida albicans adhesion and biofilm formation. Epidemiological studies report that approximately 70% of removable denture wearers suffer from denture stomatitis. Candida albicans adhesion and biofilm formation are regarded as essential prerequisites for denture stomatitis. The bioactivity and biomass of C. albicans biofilm, which was incubated in a series of twofold dilutions of nano-silver suspension at 37°C for 24 h, were determined using XTT reduction and crystal violet assays, respectively. The denture base resin specimens containing nano-silver were then used in C. albicans adhesion (37°C; 90 min; n = 9) and biofilm formation assays (37°C; 72 h; n = 9). Confocal laser scanning microscopy was used to evaluate the architectural properties of average thickness and live/dead cell ratio in the different biofilm stages that developed on the specimens. The bioactivity and biomass of C. albicans biofilm successively decreased with increasing nano-silver solution concentration. Denture base resin containing nano-silver had no effect on adhesion at low concentrations, but it exhibited anti-adhesion activity at a high concentration (5%). For 72 h biofilm formed on the resin specimens, the thickness and live/dead cell ratio were successively reduced with increasing nano-silver concentrations. Nano-silver had antifungal activity and inhibited C. albicans biofilm formation. Antifungal activity and an inhibitory effect on adhesion and biofilm formation by denture base resin containing nano-silver were discovered, especially at a higher concentration.

Dietary carbohydrates and polyols affect the microbial colonization of oral surfaces by modulating adhesion and biofilm formation. The aim of this study was to evaluate the influence of a select group of l-carbohydrates and polyols on either Streptococcus mutans or Candida albicans adhesion and biofilm formation in vitro. S. mutans or C. albicans suspensions were inoculated on polystyrene substrata in the presence of Tryptic soy broth containing 5% of the following compounds: d-glucose, d-mannose, l-glucose, l-mannose, d- and l-glucose (raceme), d- and l-mannose (raceme), l-glucose and l-mannose, sorbitol, mannitol, and xylitol. Microbial adhesion (2 h) and biofilm formation (24 h) were evaluated using MTT-test and Scanning Electron Microscopy (SEM). Xylitol and l-carbohydrates induced the lowest adhesion and biofilm formation in both the tested species, while sorbitol and mannitol did not promote C. albicans biofilm formation. Higher adhesion and biofilm formation was noted in both organisms in the presence of d-carbohydrates relative to their l-carbohydrate counterparts. These results elucidate, hitherto undescribed, interactions of the individually tested strains with l- and d-carbohydrates, and how they impact fungal and bacterial colonization. In translational terms, our data raise the possibility of using l-form of carbohydrates and xylitol for dietary control of oral plaque biofilms.

The cell wall proteins of Candida albicans play a key role in morphogenesis and pathogenesis and might be potential target sites for new specific antifungal drugs. However, these proteins are difficult to analyze because of their high heterogeneity, interconnections with wall polysaccharides (mannan, glucan, and chitin), low abundance, low solubility, and hydrophobic nature. Here we report a subproteomic approach for the study of the cell wall proteins (CWPs) from C. albicans yeast and hyphal forms. Most of the mannoproteins present in this compartment were extracted by cell wall fractionation according to the type of interactions that they establish with other structural components. CWPs were solubilized from isolated cell walls by hot SDS and dithiothreitol treatment followed by extraction either by mild alkali conditions or by enzymatic treatment with glucanases and chitinases. These highly enriched cell wall fractions were analyzed by two-dimensional PAGE, showing that a large number of proteins are involved in cell wall construction and that the wall remodeling that occurs during germ tube formation is related to changes in the composition of CWPs. We suggest that the CWP-chitin linkage is an important retention mechanism of CWPs in C. albicans mycelial forms. This article also highlights the usefulness of the combination of sequential fractionation and two-dimensional PAGE followed by Western blotting using specific antibodies against known CWPs in the characterization of incorporation mechanisms of such CWPs into the cell wall and of their interactions with other wall components. Mass spectrometry analyses have allowed the identification of several cell surface proteins classically associated with both the cell wall and other compartments. The physiological significance of the dual location of these moonlighting proteins is also discussed. This approach is therefore a powerful tool for obtaining a comprehensive and integrated view of the cell wall proteome.

Resistance to antifungal agents is a major public health concern since multidrug resistant (MDR) strains of Candida albicans have caused severe infections among immunosuppressed, diabetic and other hospital patients. This study focused on evaluating the effects of a bioactive extract (BEx) produced by a novel Streptomyces species on C. albicans cell germination and biofilm formation. Agar disk diffusion assays were used to select a streptomycete with inhibitory activity over C. albicans cells. Thereafter, minimal inhibition concentration (MIC) and time-kill values were obtained for the BEx prepared from the isolate GCAL-25. Also, the effects of BEx on biofilm formation were analyzed. Results showed that the GCAL-25 isolate from the Streptomyces genus displayed inhibitory activity on C. albicans . A paper disk soaked with BEx showed an inhibitory halo around confluent growing cells of C. albicans . The calculated MIC values for BEx indicated that C. albicans was three times more susceptible to BEx than the control fungicide, amphotericin B (AmpB). Time-kill studies with ½x and 1xMIC of BEx showed severe negative effects on cell viability, suggesting a strong fungicidal activity. In addition, an important reduction of C. albicans biofilm formation was observed. The BEx from Streptomyces sp. GCAL-25 altered yeast-to-hyphae transitions and induced abnormal cell morphology (e.g. cell shrinkage), including impairments of cell membrane integrity with negative effects on biofilm formation. https://doi.org/10.2298/ABS170908057C Received: September 8, 2017; Revised: November 29, 2017; Accepted: December 7, 2017; Published online: December 29, 2017 How to cite this article: Cordova-Davalos LE, Escobedo-Chavez KG, Evangelista-Martinez Z. Inhibition of Candida albicans cell growth and biofilm formation by a bioactive extract produced by soil Streptomyces strain GCAL-25. Arch Biol Sci. 2018;70(2):387-96.

Optimization of Candida albicans growth and biofilm formation is essential for understanding the recalcitrance of this pathogen to advance functional analysis on hospital tools and material surfaces. Optimization and quantification of biofilm have always been a challenge using the conventional one variable at a time (OVAT) method. The present study uses central composite design-based response surface methodology for optimization of conditions to induce growth and biofilm formation in Candida albicans on polystyrene microtiter plates. Statistical software package, Stat Soft®, STASTICA version 12.6 was used for data analysis. The variables considered in the design matrix were media pH, temperature, incubation period, shaker speed and inoculum size. A four-pronged quantification approach with XTT assay (cell viability), crystal violet assay (biofilm), calcofluor white assay and wet/dry weight measurements (cell mass) was used to understand different aspects of biofilm formation. Cell viability and cell mass were inversely related; however, biofilm was independent of these two factors. The study also highlighted the fact that foetal bovine serum does not significantly contribute to cell adhesion and in turn in vitro biofilm formation in some of the cultures. A high-throughput optimization of C. albicans growth and biofilm formation on polystyrene microplate has been developed and validated. This is a first time approach to optimize the interaction of parameters for C. albicans biofilm formation using RSM. Heterogeneity in growth conditions for local strains of C. albicans clinical isolates was observed. This microtiter plate-based method can be used for future screening of therapeutics for the control of C. albicans.

This study aimed to investigate the effect of ozone ultrafine bubble water (OUFBW) on the formation and growth of Candida albicans (C. albicans) biofilms and surface properties of denture base resins. OUFBWs were prepared under concentrations of 6 (OUFBW6), 9 (OUFBW9), and 11 ppm (OUFBW11). Phosphate buffered saline and ozone-free electrolyte aqueous solutions (OFEAS) were used as controls. Acrylic resin discs were made according to manufacturer instructions, and C. albicans was initially cultured on the discs for 1.5 h. A colony forming unit (CFU) assay was performed by soaking the discs in OUFBW for 5 min after forming a 24-h C. albicans biofilm. The discs after initial attachment for 1.5 h were immersed in OUFBW and then cultured for 0, 3, and 5 h. CFUs were subsequently evaluated at each time point. Moreover, a viability assay, scanning electron microscopy (SEM), Alamar Blue assay, and quantitative real-time polymerase chain reaction (qRT-PCR) test were performed. To investigate the long-term effects of OUFBW on acrylic resin surface properties, Vickers hardness (VH) and surface roughness (Ra) were measured. We found that OUFBW9 and OUFBW11 significantly degraded the formed 24-h biofilm. The time point CFU assay showed that C. albicans biofilm formation was significantly inhibited due to OUFBW11 exposure. Interestingly, fluorescence microscopy revealed that almost living cells were observed in all groups. In SEM images, the OUFBW group had lesser number of fungi and the amount of non-three-dimensional biofilm than the control group. In the Alamar Blue assay, OUFBW11 was found to suppress Candida metabolic function. The qRT-PCR test showed that OUFBW down-regulated ALS1 and ALS3 expression regarding cell-cell, cell-material adhesion, and biofilm formation. Additionally, VH and Ra were not significantly different between the two groups. Overall, our data suggest that OUFBW suppressed C. albicans growth and biofilm formation on polymethyl methacrylate without impairing surface properties.

Surfactin is a type of cyclic lipopeptide biosurfactant implicated in a wide range of applications. Although its antimicrobial activity has been characterized, its effect on Candida albicans physiology remains to be elucidated. The present study evaluated the influence of surfactin-C15 (SF) and its complexes with divalent counterions on C. albicans biofilm formation and preformed biofilms. The SF and metal(II)-SF complexes inhibited biofilm formation and reduced the metabolic activity of mature biofilms in a concentration-dependent manner. The same concentrations of the compounds studied dislodged preexisting biofilms grown on polystyrene plates. Moreover, SF and its metal(II) complexes reduced the mRNA expression of hypha-specific genes HWP1, ALS1, ALS3, ECE1 and SAP4 without exhibiting significant growth inhibition. Further research showed that the compounds tested reduced cellular surface hydrophobicity (CSH). These results suggest that SF and metal(II)-SF complexes could be used as anti-biofilm agents against C. albicans hypha-related infections in clinical practice.

SummaryCertain pathogenic bacteria and yeast form biofilms on biotic and abiotic surfaces including medical devices and implants. Hence, the development of antibiofilm coating materials becomes relevant. The virulence of those colonizing pathogens can be reduced by inhibiting biofilm formation rather than killing pathogens using excessive amounts of antimicrobials, which is touted as one of the main reasons for the development of drug resistance. Candida albicans is an opportunistic fungal pathogen, and the transition of yeast cells to hyphal cells is believed to be a crucial virulence factor. Previous studies have shown that indole and its derivatives possess antivirulence properties against various bacterial pathogens. In this study, we used various indole derivatives to investigate biofilm‐inhibiting activity against C. albicans. Our study revealed that 7‐benzyloxyindole, 4‐fluoroindole and 5‐iodoindole effectively inhibited biofilm formation compared to the antifungal agent fluconazole. Particularly, 7‐benzyloxyindole at 0.02 mM (4.5 μg ml−1) significantly reduced C. albicans biofilm formation, but had no effect on planktonic cells, and this finding was confirmed by a 2,3‐bis‐(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide (XTT) assay and three‐dimensional confocal laser scanning microscopy. Scanning electron microscopy analyses revealed that 7‐benzyloxyindole effectively inhibited hyphal formation, which explains biofilm inhibition. Transcriptomic analysis showed that 7‐benzyloxyindole downregulated the expressions of several hypha/biofilm‐related genes (ALS3,ECE1,HWP1 and RBT1). A C. albicans‐infected Caenorhabditis elegans model system was used to confirm the antivirulence efficacy of 7‐benzyloxyindole.

Biofilm formation of Candida albicans on implant overdenture materials and its removal

Many strains of Acinetobacter baumannii have been described as being able to form biofilm. Small non-coding RNAs (sRNAs) control gene expression in many regulatory circuits in bacteria. The aim of the present work was to provide a global description of the sRNAs produced both by planktonic and biofilm-associated (sessile) cells of A. baumannii ATCC 17978, and to compare the corresponding gene expression profiles to identify sRNAs molecules associated to biofilm formation and virulence. sRNA was extracted from both planktonic and sessile cells and reverse transcribed. cDNA was subjected to 454-pyrosequencing using the GS-FLX Titanium chemistry. The global analysis of the small RNA transcriptome revealed different sRNA expression patterns in planktonic and biofilm associated cells, with some of the transcripts only expressed or repressed in sessile bacteria. A total of 255 sRNAs were detected, with 185 of them differentially expressed in the different types of cells. A total of 9 sRNAs were expressed only in biofilm cells, while the expression of other 21 coding regions were repressed only in biofilm cells. Strikingly, the expression level of the sRNA 13573 was 120 times higher in biofilms than in planktonic cells, an observation that prompted us to further investigate the biological role of this non-coding transcript. Analyses of an isogenic mutant and over-expressing strains revealed that the sRNA 13573 gene is involved in biofilm formation and attachment to A549 human alveolar epithelial cells. The present work serves as a basis for future studies examining the complex regulatory network that regulate biofilm biogenesis and attachment to eukaryotic cells in A. baumannii ATCC 17978.

Targeting hypoxia: the role of ethanolic extract of Coptis chinensis Franch. in modulating HIF-1α/IL-17 axis in oropharyngeal candidiasis.

This study aimed to address the significant problems of bacterial biofilms found in medical fields and many industries. It explores the potential of classic photoactive carbon dots (CDots), with 2,2′-(ethylenedioxy)bis (ethylamine) (EDA) for dot surface functionalization (thus, EDA-CDots) for their inhibitory effect on B. subtilis biofilm formation and the inactivation of B. subtilis cells within established biofilm. The EDA-CDots were synthesized by chemical functionalization of selected small carbon nanoparticles with EDA molecules in amidation reactions. The inhibitory efficacy of CDots with visible light against biofilm formation was dependent significantly on the time point when CDots were added; the earlier the CDots were added, the better the inhibitory effect on the biofilm formation. The evaluation of antibacterial action of light-activated EDA-CDots against planktonic B. subtilis cells versus the cells in biofilm indicate that CDots are highly effective for inactivating planktonic cells but barely inactivate cells in established biofilms. However, when coupling with chelating agents (e.g., EDTA) to target the biofilm architecture by breaking or weakening the EPS protection, much enhanced photoinactivation of biofilm-associated cells by CDots was achieved. The study demonstrates the potential of CDots to prevent the initiation of biofilm formation and to inhibit biofilm growth at an early stage. Strategic combination treatment could enhance the effectiveness of photoinactivation by CDots to biofilm-associated cells.

Introduction: Candida albicans (C. albicans) is the most common fungus found in the human oral cavity. This fungus has the ability to form a biofilm that causes infectious diseases in the oral cavity. Nowadays, the incidence of infectious diseases caused by C.albicans was increasing due to resistance to antifungal drugs. This study aimed to investigate the effect of Moringa oleifera ethyl acetate extract on the inhibition of C. albicans planktonic cell growth and biofilm formation in vitro. Methods: C. albicans (ATCC 10231) was the fungus used in this study. Determination of inhibition planktonic cell growth by microdilution method. The polystyrene microplate assay method was used to test the inhibition of C. albicans biofilm formation. The extract concentrations used in this study were 25%, 12.5%, 6.25%, 3.13%, and 1.57%, respectively. A crystal violet (CV) assay assessed the biofilm's inhibitory activity. Results: The minimal inhibitory concentration of Moringa oleifera leaf ethyl acetate extract against the planktonic form of C.albicans was found to be 1.57%. Starting at 6.25% concentration, Moringa leaf ethyl acetate extract inhibits the formation of C. albicans biofilm. Conclusion: Since Moringa oleifera leaf ethyl acetate extract inhibits C. albicans planktonic and biofilm formation, it has the potential to be developed as an alternative anti-fungal agent.&#x0D; Keywords: Moringa leaf extract, planktonic cells, biofilm, Candida albicans

Resistance caused by the formation of the Candida albicans (C. albicans) biofilm is one of the main reasons for antifungal therapy failure. Thus, it is important to find indicators that predict C. albicans biofilm formation to provide evidence for the early prevention and treatment of the C. albicans biofilms. In this study, C. albicans samples were selected from C. albicans septicemia that were sensitive to common antifungal agents. It was found that the agglutinin-like sequence 3 (ALS3) gene was differentially expressed in free, antifungal, drug-sensitive C. albicans. The average ALS3 gene expression was higher in the C. albicans strains with biofilm formation than that in the C. albicans strains without biofilm formation. Then, it was further confirmed that the rate of biofilm formation was higher in the high ALS3 gene expression group than that in the low ALS3 gene expression group. It was found that C. albicans with biofilm formation was more resistant to fluconazole, voriconazole, and itraconazole. However, it maintained its sensitivity to caspofungin and micafungin in vitro and in mice. Further experiments regarding the prevention of C. albicans biofilm formation were performed in mice, in which only caspofungin and micafungin prevented C. albicans biofilm formation. These results suggest that the expression level of ALS3 in C. albicans may be used as an indicator to determine whether C. albicans will form biofilms. The results also show that the biofilm formation of C. albicans remained sensitive to caspofungin and micafungin, which may help to guide the selection of clinical antifungal agents for prevention and therapy.