Characteristics of biofilm formation by Candida albicans

A variety of manifestations of Candida albicans infections are associated with the formation of biofilms on the surface of biomaterials. Cells in biofilms display phenotypic traits that are dramatically different from their free-floating planktonic counterparts, such as increased resistance to anti-microbial agents and protection form host defenses. Here, we describe the characteristics of C. albicans biofilm development using a 96 well microtitre plate model, microscopic observations and a colorimetric method based on the use of a modified tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide, XTT) to monitor metabolic activities of cells within the biofilm. C. albicans biofilm formation was characterized by initial adherence of yeast cells (0-2 h), followed by germination and micro-colony formation (2-4 h), filamentation (4-6 h), monolayer development (6-8 h), proliferation (8-24 h) and maturation (24-48 h). The XTT-reduction assay showed a linear relationship between cellular density of the biofilm and metabolic activity. Serum and saliva pre-conditioning films increased the initial attachment of C. albicans, but had minimal effect on subsequent biofilm formation. Scanning electron microscopy and confocal scanning laser microscopy were used to visualize C. albicans biofilms. Mature C. albicans biofilms consisted of a dense network of yeasts cells and hyphal elements embedded within exopolymeric material. C. albicans biofilms displayed a complex three dimensional structure which demonstrated spatial heterogeneity and a typical architecture showing microcolonies with ramifying water channels. Antifungal susceptibility testing demonstrated the increased resistance of sessile C. albicans cells against clinically used fluconazole and amphotericin B as compared to their planktonic counterparts.     

Candida albicans biofilm formation on peptide functionalized polydimethylsiloxane

In order to prevent biofilm formation by Candida albicans, several cationic peptides were covalently bound to polydimethylsiloxane (PDMS). The salivary peptide histatin 5 and two synthetic variants (Dhvar 4 and Dhvar 5) were used to prepare peptide functionalized PDMS using 4-azido-2,3,5,6-tetrafluoro-benzoic acid (AFB) as an interlinkage molecule. In addition, polylysine-, polyarginine-, and polyhistidine-PDMS surfaces were prepared. Dhvar 4 functionalized PDMS yielded the highest reduction of the number of C. albicans biofilm cells in the Modified Robbins Device. Amino acid analysis demonstrated that the amount of peptide immobilized on the modified disks was in the nanomole range. Poly-d-lysine PDMS, in particular the homopeptides with low molecular weight (2500 and 9600) showed the highest activity against C. albicans biofilms, with reductions of 93% and 91%, respectively. The results indicate that the reductions are peptide dependent.     

Inhibition of Candida albicans biofilm formation by farnesol,a quorum-sensing molecule

Farnesol is a quorum-sensing molecule that inhibits filamentation in Candida albicans. Both filamentation and quorum sensing are deemed to be important factors in C. albicans biofilm development. Here we examined the effect of farnesol on C. albicans biofilm formation. C. albicans adherent cell populations (after 0, 1, 2, and 4 h of adherence) and preformed biofilms (24 h) were treated with various concentrations of farnesol (0, 3, 30, and 300 micro M) and incubated at 37 degrees C for 24 h. The extent and characteristics of biofilm formation were then assessed microscopically and with a semiquantitative colorimetric technique based on the use of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide. The results indicated that the effect of farnesol was dependent on the concentration of this compound and the initial adherence time, and preincubation with 300 micro M farnesol completely inhibited biofilm formation. Supernatant media recovered from mature biofilms inhibited the ability of planktonic C. albicans to form filaments, indicating that a morphogenetic autoregulatory compound is produced in situ in biofilms. Northern blot analysis of RNA extracted from cells in biofilms indicated that the levels of expression of HWP1, encoding a hypha-specific wall protein, were decreased in farnesol-treated biofilms compared to the levels in controls. Our results indicate that farnesol acts as a naturally occurring quorum-sensing molecule which inhibits biofilm formation, and we discuss its potential for further development and use as a novel therapeutic agent.     

Lipopeptides from Bacillus strain AR2 inhibits biofilm formation by Candida albicans

The ability of the human fungal pathogen Candida albicans to reversibly switch between different morphological forms and establish biofilms is crucial for establishing infection. Targeting phenotypic plasticity and biofilm formation in C. albicans represents a new concept for antifungal drug discovery. The present study evaluated the influence of cyclic lipopeptide biosurfactant produced by Bacillus amyloliquefaciens strain AR2 on C. albicans biofilms. The biosurfactant was characterized as a mixture of iturin and fengycin by MALDI-TOF and amino acid analysis. The biosurfactant exhibited concentration dependent growth inhibition and fungicidal activity. The biosurfactant at sub-minimum growth inhibition concentration decreased cell surface hydrophobicity, hindered germ tube formation and reduced the mRNA expression of hyphae-specific gene HWP1 and ALS3 without exhibiting significant growth inhibition. The biosurfactants inhibited biofilm formation in the range of 46–100 % depending upon the concentration and Candida strains. The biosurfactant treatment dislodged 25–100 % of preformed biofilm from polystyrene plates. The biosurfactant retained its antifungal and antibiofilm activity even after exposure to extreme temperature. By virtue of the ability to inhibit germ tube and biofilm formation, two important traits of C. albicans involved in establishing infection, lipopeptides from strain AR2 may represent a potential candidate for developing heat stable anti-Candida drugs.     

Transcriptional profiling of the early stages of germination in Candida albicans by real-time RT-PCR

By using real-time RT-PCR, we profiled the expression of CGR1, CaMSI3, EFG1, NRG1, and TUP1 in Candida albicans strains JCM9061 and CAI4 under several conditions, including induction of morphological transition, heat shock, and treatment with calcium inhibitors. Expression of CaMSI3 changed under these growth conditions except during heat shock. CGR1 expression increased during the early stages of hyphal growth in JCM9061, while expression was strain-dependent during heat shock. Both EFG1 and NRG1 were similarly expressed under hypha-inducing conditions and heat shock. Expression of TUP1 was slightly different from the expression of EFG1 or NRG1.     

Genome-wide transcriptional profiling of the cyclic AMP-dependent signaling pathway during morphogenic transitions of Candida albicans

Candida albicans is an opportunistic human fungal pathogen that causes systemic candidiasis as well as superficial mucosal candidiasis. In response to the host environment, C. albicans transitions between yeast and hyphal forms. In particular, hyphal growth is important in facilitating adhesion and invasion of host tissues, concomitant with the expression of various hypha-specific virulence factors. In previous work, we showed that the cyclic AMP (cAMP) signaling pathway plays a crucial role in morphogenic transitions and virulence of C. albicans by studying genes encoding adenylate cyclase-associated protein (CAP1) and high-affinity phosphodiesterase (PDE2) (Y. S. Bahn, J. Staab, and P. Sundstrom, Mol. Microbiol. 50:391-409, 2003; and Y. S. Bahn and P. Sundstrom, J. Bacteriol. 183:3211-3223, 2001). However, little is known about the downstream targets of the cAMP signaling pathway that are responsible for morphological transitions and the expression of virulence factors. Here, microarrays were probed with RNA from strains with hypoactive (cap1/cap1 null mutant), hyperactive (pde2/pde2 null mutant), and wild-type cAMP signaling pathways to provide insight into the molecular mechanisms of virulence that are regulated by cAMP and that are related to the morphogenesis of C. albicans. Genes controlling metabolic specialization, cell wall structure, ergosterol/lipid biosynthesis, and stress responses were modulated by cAMP during hypha formation. Phenotypic traits predicted to be regulated by cAMP from the profiling results correlated with the relative strengths of the mutants when tested for resistance to azoles and subjected to heat shock stress and oxidative/nitrosative stress. The results from this study provide important insights into the role of the cAMP signaling pathway not only in morphogenic transitions of C. albicans but also for adaptation to stress and for survival during host infections.     

抗白念珠菌生物被膜药物的研究进展

白念珠菌是临床最常见的一种能产生生物被膜的致病真菌,所产生的生物被膜是导致高度耐药性和临床白念珠菌反复感染的直接原因.近年来,科学家们开始关注天然产物的抗生物被膜活性,以及不同药物联合应用的抗生物被膜效果,该文对抗白念珠菌生物被膜药物的研究进展作一综述.     

Requirement for Candida albicans Sun41 in biofilm formation and virulence

The cell wall of Candida albicans lies at the crossroads of pathogenicity and therapeutics. It contributes to pathogenicity through adherence and invasion; it is the target of both chemical and immunological antifungal strategies. We have initiated a dissection of cell wall function through targeted insertional mutagenesis of cell wall-related genes. Among 25 such genes, we were unable to generate homozygous mutations in 4, and they may be essential for viability. We created homozygous mutations in the remaining 21 genes. Insertion mutations in SUN41, Orf19.5412, Orf19.1277, MSB2, Orf19.3869, and WSC1 caused hypersensitivity to the cell wall inhibitor caspofungin, while two different ecm33 insertions caused mild caspofungin resistance. Insertion mutations in SUN41 and Orf19.5412 caused biofilm defects. Through analysis of homozygous sun41Delta/sun41Delta deletion mutants and sun41Delta/sun41Delta+pSUN41-complemented strains, we verified that Sun41 is required for biofilm formation and normal caspofungin tolerance. The sun41Delta/sun41Delta mutant had altered expression of four cell wall damage response genes, thus suggesting that it suffers a cell wall structural defect. Sun41 is required for inducing disease, because the mutant was severely attenuated in mouse models of disseminated and oropharyngeal candidiasis. Although the mutant produced aberrant hyphae, it had no defect in damaging endothelial or epithelial cells, unlike many other hypha-defective mutants. We suggest that the sun41Delta/sun41Delta cell wall defect is the primary cause of its attenuated virulence. As a small fungal surface protein with predicted glucosidase activity, Sun41 represents a promising therapeutic target.     

A Candida albicans early stage biofilm detachment event in rich medium

Background  

Dispersal from Candida albicans biofilms that colonize catheters is implicated as a primary factor in the link between contaminated catheters and life threatening blood stream infections (BSI). Appropriate in vitro C. albicans biofilm models are needed to probe factors that induce detachment events.     

Environmental pH modulates biofilm formation and matrix composition in Candida albicans and Candida glabrata

Abstract

Candida species are fungal opportunistic pathogens capable of colonizing and infecting various human anatomical sites, where they have to adapt to distinct niche-specific pH conditions. The aim of this study was to analyse the features of Candida albicans and Candida glabrata biofilms developed under neutral and vaginal acidic (pH 4) conditions. C. albicans produced thicker and more filamentous biofilms under neutral than under acidic conditions. On the other hand, the formation of biofilms by C. glabrata was potentiated by the acidic conditions suggesting the high adaptability of this species to the vaginal environment. In general, both species developed biofilms containing higher amounts of matrix components (protein and carbohydrate) under neutral than acidic conditions, although the opposite result was found for one C. glabrata strain. Overall, this study contributes to a better understanding of the modulation of C. albicans and C. glabrata virulence by specific pH conditions.     

In vivo inhibitory effect on the biofilm formation of Candida albicans by liverwort derived riccardin D

Riccardin D, a macrocyclic bisbibenzyl isolated from Chinese liverwort Dumortiera hirsute, has been proved to have inhibitory effect on biofilms formation of Candida albicans in in vitro study. Our present study aims to investigate the in vivo effect and mechanisms of riccardin D against C. albicans biofilms when used alone or in combination with clinical using antifungal agent fluconazole. XTT reduction assay revealed riccardin D had both prophylactic and therapeutic effect against C. albicans biofilms formation in a dose-dependent manner when using a central venous catheter related infective animal model. Scanning electron microscope and laser confocal scanning microscope showed that the morphology of biofilms was altered remarkably after riccardin D treatment, especially hypha growth inhibition. To uncover the underlying molecular mechanisms, quantitative real-time RT-PCR was performed to observe the variation of related genes. The downregulation of hypha-specific genes such as ALS1, ALS3, ECE1, EFG1, HWP1 and CDC35 following riccardin D treatment suggested riccardin D inhibited the Ras-cAMP-Efg pathway to retard the hypha formation, then leading to the defect of biofilms maturation. Moreover, riccardin D displayed an increased antifungal activity when administered in combination with fluconazole. Our study provides a potential clinical application to eliminate the biofilms of relevant pathogens.     

Efficacy of surface-generated nitric oxide against Candida albicans adhesion and biofilm formation

This report details the efficacy of nitric oxide (NO)-releasing xerogel surfaces composed of N-(6-aminohexyl)aminopropyl trimethoxysilane (AHAP3) and isobutyltrimethoxysilane (BTMOS) against Candida albicans adhesion, viability, and biofilm formation. A parallel plate flow cell assay was used to examine the effect of NO on planktonic fungal cells. Nitric oxide fluxes as low as 14 pmol cm^?2 s^?1 were sufficient to reduce fungal adhesion by ～49% over the controls after 90 min. By utilizing a fluorescence live/dead assay and replicate plating, NO flux was determined to reduce fungal viability in a dose-dependent manner. The formation of C. albicans biofilms on NO-releasing xerogel-coated silicon rubber (SiR) coupons was impeded when compared to control (non-NO-releasing) and bare SiR surfaces. The synergistic efficacy of NO and silver sulfadiazine against adhered fungal cells and biofilms is reported with increased killing and biofilm inhibition over NO alone.     

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.     

Genome-wide single-nucleotide polymorphism map for Candida albicans

Single-nucleotide polymorphisms (SNPs) are essential tools for studying a variety of organismal properties and processes, such as recombination, chromosomal dynamics, and genome rearrangement. This paper describes the development of a genome-wide SNP map for Candida albicans to study mitotic recombination and chromosome loss. C. albicans is a diploid yeast which propagates primarily by clonal mitotic division. It is the leading fungal pathogen that causes infections in humans, ranging from mild superficial lesions in healthy individuals to severe, life-threatening diseases in patients with suppressed immune systems. The SNP map contains 150 marker sequences comprising 561 SNPs and 9 insertions-deletions. Of the 561 SNPs, 437 were transition events while 126 were transversion events, yielding a transition-to-transversion ratio of 3:1, as expected for a neutral accumulation of mutations. The average SNP frequency for our data set was 1 SNP per 83 bp. The map has one marker placed every 111 kb, on average, across the 16-Mb genome. For marker sequences located partially or completely within coding regions, most contained one or more nonsynonymous substitutions. Using the SNP markers, we identified a loss of heterozygosity over large chromosomal fragments in strains of C. albicans that are frequently used for gene manipulation experiments. The SNP map will be useful for understanding the role of heterozygosity and genome rearrangement in the response of C. albicans to host environments.     

Candida albicans biofilm formation on soft denture liners and efficacy of cleaning protocols

doi: 10.1111/j.1741‐2358.2011.00485.x
Candida albicans biofilm formation on soft denture liners and efficacy of cleaning protocols Objective: The aim of this study was to investigate Candida albicans biofilm formation on denture liners and to analyse the efficacy of cleaning protocols. Material and methods: Specimens were prepared from four silicone‐based soft denture liners. After artificial ageing and surface free energy determination, specimens were incubated with saliva (2 h) and Candida albicans ATCC 10231 for either short‐ (2.5 h) or long‐term (24 h) biofilm formation. Adherent cells were determined either after incubation of specimens with Candida albicans or after treatment with different denture cleaning protocols. Statistical analysis was performed using one‐way anova and the Games–Howell test (α = 0.05). Results: For both short‐ and long‐term biofilm formation, similar amounts of Candida albicans cells were found on the surface of the different liners (p = 0.295 and 0.178, respectively). For both short‐ and long‐term biofilm formation, the highest cleaning efficacy was observed for sodium hypochlorite (NaOCl; p < 0.01). The efficacy of the chemical denture cleaner in removing long‐term Candida albicans biofilms was significantly lower than the efficacy of removal by brushing (p < 0.001). Conclusion: Different silicone‐based soft denture liners yield similar Candida albicans biofilm formation on their surface. The highest efficacy for the removal of Candida albicans biofilms was identified for NaOCl. Chemical denture cleaners appear to have rather low efficacy to remove mature Candida albicans biofilms.     

Cyclooxygenase inhibitors reduce biofilm formation and yeast-hypha conversion of fluconazole resistant Candida albicans

The incidence of fluconazole-resistant Candida albicans has been increasing worldwide. Both biofilm and fungal morphogenesis are main virulence factors of C. albicans cells. Extracellular fungal prostaglandins are synthesized during biofilm adhesion and development and through yeast-hypha conversion. Hence, we targeted prostaglandin synthesis with various cyclooxygenase (COX) inhibitors (aspirin, diclofenac, ketoprofen, tenoxicam, and ketorolac) and assessed their effect on fungal adhesion, biofilm formation, and yeast-hypha conversion in clinical isolates of Fluconazole resistant C. albicans. Significant reduction in fungal adhesion and detachment of mature biofilm was attained down to 1 mM concentrations of anti-inflammatory agents. Microscopical examination of fungal cells in the presence of the tested drugs showed significant reduction of germ tube formation. Therefore, COX inhibitors have a significant effect on reduction of Candida adhesion and biofilm development in correlation with fungal morphogenesis. Moreover, inhibition of C. albicans by COX inhibitors gave synergistic activity with fluconazole suggesting that combination therapeutic strategies may be fruitful for management of infection of Fluconazole resistant C. albicans.