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.     

壳聚糖体外抗白念珠菌生物膜作用的初步研究

目的观察壳聚糖对白念珠菌生物膜形成的影响,探讨其可能的作用机制。方法 XTT减低法评价壳聚糖对白念珠菌生物膜形成及黏附的影响,镜下观察壳聚糖对白念珠菌生物膜形态的影响;实时定量RT-PCR法观察壳聚糖对白念珠菌的Ras信号通路因子CDC35、PDE2、EFG1和HWP1的基因表达的影响。结果低浓度(0.02 mg/mL)和高浓度(0.32mg/mL)壳聚糖对白念珠菌生物膜形成的抑制率分别为(19.6±1.2)%和(96.96±0.6)%,0.16 mg/mL浓度下壳聚糖对早期(0 h)、中期(12 h)和成熟期(48 h)的生物膜抑制率分别为(78.6±0.5)%、(54.4±0.9)%和(41.1±1.1)%,不同浓度的壳聚糖对各黏附阶段的白念珠菌细胞黏附均有抑制作用,壳聚糖可剂量依赖性地下调白念珠菌生物膜Ras信号通路基因CDC35、EFG1和HWP1的表达水平,上调Ras信号通路抑制剂PDE2的基因表达水平(P<0.05)。结论壳聚糖可能通过影响Ras信号通路及抑制细胞黏附而对白念珠菌生物膜的形成具有抑制作用。     

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.     

The antagonistic effect of Saccharomyces boulardii on Candida albicans filamentation, adhesion and biofilm formation

The dimorphic fungus Candida albicans is a member of the normal flora residing in the intestinal tract of humans. In spite of this, under certain conditions it can induce both superficial and serious systemic diseases, as well as be the cause of gastrointestinal infections. Saccharomyces boulardii is a yeast strain that has been shown to have applications in the prevention and treatment of intestinal infections caused by bacterial pathogens. The purpose of this study was to determine whether S. boulardii affects the virulence factors of C. albicans . We demonstrate the inhibitory effect of live S. boulardii cells on the filamentation (hyphae and pseudohyphae formation) of C. albicans SC5314 strain proportional to the amount of S. boulardii added. An extract from S. boulardii culture has a similar effect. Live S. boulardii and the extract from S. boulardii culture filtrate diminish C. albicans adhesion to and subsequent biofilm formation on polystyrene surfaces under both aerobic and microaerophilic conditions. This effect is very strong and requires lower doses of S. boulardii cells or concentrations of the extract than serum-induced filamentation tests. Saccharomyces boulardii has a strong negative effect on very important virulence factors of C. albicans , i.e. the ability to form filaments and to adhere and form biofilms on plastic surfaces.     

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.     

白念珠菌生物膜体内外模型研究进展

白念珠菌引起的慢性难治性感染与其形成的生物膜密切相关,生物膜的诸多生物学特征明显不同于游离菌.合适的生物膜模型的构建对于研究白念珠菌生物膜的形成机制以及药物干预至关重要.该文结合作者自身的研究,对目前国内外关于白念珠菌生物膜体外、体内多种模型的构建方法作一综述.     

CRK1基因缺失对白念珠菌形态、黏附、生物被膜的影响

目的 研究CRK1基因缺失对白念珠菌形态、黏附、生物被膜的影响.方法 显微镜下观察,计算菌丝形成率,比较CRK1基因缺失菌（Δcrk1菌）及标准菌SC5314形成菌丝的能力;建立肠黏膜模型,计算黏附率,评价CRK1基因缺失对白念珠菌黏附的影响;MTT法及结晶紫法（CV）评价CRK1基因缺失对白念珠菌生物被膜形成的影响.结果 与SC5314相比,Δcrk1菌分别在10％胎牛血清和RPMI-1640培养条件下形成菌丝能力均较弱,两者之间有统计学差异;Δcrk1菌在60、90、120 min时对肠黏膜的黏附数明显少于标准菌SC5314,两者之间有统计学差异;通过MTT法、结晶紫法两种方法证实了,在经48 h培养后,Δcrk1菌与其标准菌SC5314相比,形成生物膜的能力弱,两者之间差异有统计学差异.结论 CRK 1基因缺失影响白念珠菌菌丝二态性的转化,进而影响黏附力和生物被膜的形成.     

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.     

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.     

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.     

黄芩素与氟康唑协同抗白念珠菌生物被膜作用研究

目的：研究黄芩素与氟康唑合用对白念珠菌生物被膜形成的影响。方法采用激光共聚焦显微镜观察黄芩素与氟康唑合用对白念珠菌生物被膜生长形态的影响;采用 XTT 法考察黄芩素与氟康唑合用对白念珠菌生物被膜形成能力的影响;应用水-烃两相测定实验考察黄芩素与氟康唑合用对白念珠菌生物被膜细胞表面疏水性（ Cell surface hydrophobicity, CSH）的影响;应用实时定量 RT-PCR（Real Time RT-PCR）实验考察黄芩素与氟康唑合用对白念珠菌 CSH1、EFG1、HWP1、ALS1基因表达的影响。结果黄芩素与氟康唑合用能够协同抑制白念珠菌生物被膜的形成,经黄芩素与氟康唑处理的白念珠菌不能形成正常的生物被膜,其生长动力学及细胞表面疏水性下降,细胞疏水性相关基 CSH1、菌丝形成调控基因EFG1、黏附相关基因 HWP1基因的表达水平降低。结论黄芩素与氟康唑合用可协同抑制白念珠菌生物被膜的形成。     

Eap1p, an adhesin that mediates Candida albicans biofilm formation in vitro and in vivo

Candida albicans is the leading cause of systemic fungal infections in immunocompromised humans. The ability to form biofilms on surfaces in the host or on implanted medical devices enhances C. albicans virulence, leading to antimicrobial resistance and providing a reservoir for infection. Biofilm formation is a complex multicellular process consisting of cell adhesion, cell growth, morphogenic switching between yeast form and filamentous states, and quorum sensing. Here we describe the role of the C. albicans EAP1 gene, which encodes a glycosylphosphatidylinositol-anchored, glucan-cross-linked cell wall protein, in adhesion and biofilm formation in vitro and in vivo. Deleting EAP1 reduced cell adhesion to polystyrene and epithelial cells in a gene dosage-dependent manner. Furthermore, EAP1 expression was required for C. albicans biofilm formation in an in vitro parallel plate flow chamber model and in an in vivo rat central venous catheter model. EAP1 expression was upregulated in biofilm-associated cells in vitro and in vivo. Our results illustrate an association between Eap1p-mediated adhesion and biofilm formation in vitro and in vivo.     

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.