J. Y. Y. Yau

Candida Species Exhibit Differential In Vitro Hemolytic Activities

ABSTRACT A total of 80 Candida isolates representing 14 species were examined for their respective responses to an in vitro hemolytic test. A modification of a previously described plate assay system where the yeasts are incubated on glucose (3%)-enriched sheep blood agar in a carbon dioxide (5%)-rich environment for 48 h was used to evaluate the hemolytic activity. A group of eight Candidaspecies which included Candida albicans (15 isolates),C. dubliniensis (2), C. kefyr(2), C. krusei (4), C. zeylanoides (1), C. glabrata(34), C. tropicalis (5), andC. lusitaniae (2) demonstrated both alpha and beta hemolysis at 48 h postinoculation. Only alpha hemolysis was detectable in four Candida species, viz., C. famata (3), C. guilliermondii(4), C. rugosa (1), and C. utilis (1), while C. parapsilosis (5) and C. pelliculosa (1) failed to demonstrate any hemolytic activity after incubation for 48 h or longer. This is the first study to demonstrate the variable expression profiles of hemolysins by different Candida species.

Water Concentration in Self-etching Primers Affects their Aggressiveness and Bonding Efficacy to Dentin

Water is required to ionize acid resin monomers for demineralization of tooth substrates. We tested the null hypothesis that altering the water concentration in two-step self-etching primers has no effect on their aggressiveness and bonding efficacy to dentin. Five experimental self-etching primers were prepared with resin-water-ethanol volume ratios of 9-0-1, 8-1-1, 7-2-1, 5-4-1, and 3-6-1. They were applied to smear-layer-covered dentin, followed by a bonding resin and composite build-ups for microtensile bond testing and TEM examination of tracer penetration. Increasing water concentration from 0–60 vol% improved acidic monomer ionization that was manifested as increasing hybrid layer thickness. However, significantly higher bond strength was observed in the 7-2-1 group, with minimal nanoleakage in the corresponding hybrid layer. When self-etching primers are formulated, a balance must be achieved to provide sufficient water for adequate ionization of the acidic monomers, without lowering the resin concentration too much, to optimize their bonding efficacy to dentin.

Interspecies variation in Candida biofilm formation studied using the Calgary biofilm device.

An in vitro assay to study multiple Candida biofilms, in parallel, has been carried out using the Calgary biofilm device (CBD). We here report: i) standardization of the CBD for Candida albicans biofilm formation, ii) kinetics of C. albicans biofilm formation, iii) biofilm formation by five Candida species, and iv) effect of dietary carbohydrates on biofilm formation. The biofilm metabolic activity on all CBD pegs was similar (p=0.6693) and C. albicans biofilm formation revealed slow growth up to 36 h and significantly higher growth up to 48 h (p<0.001). Significant differences in total biofilm metabolic activity were seen for glucose, fructose and lactose grown C. albicans compared with sucrose and maltose grown yeasts. Candida krusei developed the largest biofilm mass (p<0.05) relative to C. albicans, C. glabrata, C. dubliniensis and C. tropicalis. Scanning electron microscopy revealed that C. krusei produced a thick multilayered biofilm of pseudohyphal forms embedded within the polymer matrix, whereas C. albicans, C. dubliniensis and C. tropicalis biofilms consisted of clusters or chains of cells with sparse extracellular matrix material. We conclude that CBD is a useful, simple, low cost miniature device for parallel study of Candida biofilms and factors modulating this phenomenon.

Pseudomonas aeruginosa inhibits in-vitro Candida biofilm development

BackgroundElucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as Candida spp. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species biofilm development.ResultsA significant reduction in colony forming units (CFU) of C. parapsilosis (90 min), C. albicans and C. tropicalis (90 min, 24 h and 48 h), C. dubliniensis and C. glabrata, (24 h and 48 h) was noted when co-cultured with P. aeruginosa in comparison to their monospecies counterparts (P < 0.05). A simultaneous significant reduction in P. aeruginosa numbers grown with C. albicans (90 min and 48 h), C. krusei (90 min, 24 h and 48 h),C. glabrata, (24 h and 48 h), and an elevation of P. aeruginosa numbers co-cultured with C. tropicalis (48 h) was noted (P < 0.05). When data from all Candida spp. and P. aeruginosa were pooled, highly significant mutual inhibition of biofilm formation was noted (Candida P < 0.001, P. aeruginosa P < 0.01). Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) analyses confirmed scanty architecture in dual species biofilm in spite of dense colonization in monospecies counterparts.ConclusionsP. aeruginosa and Candida in a dual species environment mutually suppress biofilm development, both quantitatively and qualitatively. These findings provide a foundation to clarify the molecular basis of bacterial-fungal interactions, and to understand the pathobiology of mixed bacterial-fungal infections.

Differential phospholipase gene expression by Candida albicans in artificial media and cultured human oral epithelium

Phospholipases B1, B2, C and D of Candida albicans play a significant role in the host invasive process. Hence we evaluated the in vitro expression of PLB1, PLB2, PLC1 and PLD1 in phospholipase‐positive (PL+) and ‐deficient (PL−) C. albicans isolates in egg yolk agar (EYA), yeast peptone dextrose broth (YPD), and in a model of oral candidiasis based on reconstituted human oral epithelium (RHOE). The growth of Candida was then determined in YPD and its cellular invasion was investigated using the RHOE model. The PL+ group demonstrated PLB1, PLB2, PLC1 and PLD1 expression in both EYA and YPD, in contrast to the PL− group, which expressed only PLB2 and PLD1. Although PL+ isolates grew profusely in the RHOE model, they expressed only PLB2, PLC1 and PLD1, and not PLB1. Gene expression investigations could not be carried out with PL− isolates due to their inability to grow in the RHOE model. Significant growth differences in YPD medium were also observed within the PL+ and PL− groups. Taken together, these findings indicate that phospholipase gene expression in C. albicans is differentially affected by their growth milieu, and this in turn may modulate the disease outcomes in vivo.

Antifungal activity of black tea polyphenols (catechins and theaflavins) against candida species

Background/Aims: The polyphenols catechins and theaflavins in black tea have been shown to possess many medicinal properties, including anticancer activity and some antifungal characteristics, but there have been few studies of their anti-Candida activity. In this paper we report the results of our study of the anti-Candida activity of tea polyphenols. Methods: The effects of 4 different concentrations of catechins and theaflavins were evaluated on 5 isolates each of 5 Candida species employing an agar diffusion growth inhibition assay. The minimum inhibitory concentration (MIC) of the polyphenols against C. albicans was determined. The post-antifungal effect (PAFE) of the polyphenols for C. albicans was investigated. C. albicans cells exposed to polyphenols were studied using a scanning electron microscope (SEM). Results: Both polyphenols showed anti-Candida activity against all tested Candida species and demonstrated a MIC of 6.25 mg/ml for C. albicans. C. glabrata was found to be the most sensitive species followed by C. parapsilosis, C. albicans, C. krusei and C. tropicalis (p < 0.05 for all). Significant intraspecies variations in sensitivity were noted among C. parapsilosis and C. tropicalis (p < 0.001) for both polyphenols. Theaflavins displayed standard PAFE while catechins showed a paradoxical PAFE with all isolates of C. albicans. SEM revealed considerable cell wall damage of C. albicans cells exposed to the polyphenols. Conclusion: The study reveals for the first time the anti-Candida properties of black tea polyphenols that may find therapeutic applications in future.

Escherichia coli and its lipopolysaccharide modulate in vitro Candida biofilm formation

Demystification of microbial behaviour in mixed biofilms could have a major impact on our understanding of infectious diseases. The objectives of this study were to evaluate in vitro the interactions of six different Candida species and a Gram-negative coliform, Escherichia coli, in dual-species biofilms, and to assess the effect of E. coli LPS on Candida biofilm formation. A single isolate of E. coli ATCC 25922 and six different species of Candida, Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Candida tropicalis ATCC 13803, Candida parapsilosis ATCC 22019 and Candida dubliniensis MYA-646, were studied using a standard biofilm assay. Each Candida species was co-cultured with E. coli on a polystyrene surface and biofilm formation was quantified by a c.f.u. assay. The biofilm was then analysed by Live/Dead staining and fluorescence microscopy (confocal laser-scanning microscopy, CLSM), whilst scanning electron microscopy (SEM) was employed to visualize the biofilm architecture. The effect of E. coli LPS on Candida biofilm cell activity at defined time intervals was assessed with an XTT reduction assay. A significant quantitative reduction in c.f.u. counts of C. tropicalis (after 90 min), C. parapsilosis (after 90 min and 24 h), C. krusei (after 24 h) and C. dubliniensis (after 24 and 48 h) was noted on incubation with E. coli in comparison with their monospecies biofilm counterparts (P <0.05). On the other hand, a simultaneous and significant reduction in E. coli cell numbers occurred on co-culture with C. albicans (after 90 min), and an elevation of E. coli cell numbers followed co-culture with C. tropicalis (after 24 h) and C. dubliniensis (after 24 h and 48 h) (P <0.05). All quantitative findings were confirmed by SEM and CLSM analyses. By SEM observation, dual-species biofilms demonstrated scanty architecture with reduced visible cell counts at all stages of biofilm development, despite profuse growth and dense colonization in their single-species counterparts. Significantly elevated metabolic activity, as assessed by XTT readings, was observed in E. coli LPS-treated C. tropicalis and C. parapsilosis biofilms (after 48 h), whilst this had the opposite effect for C. dubliniensis (after 24 h) (P <0.05). These data indicate that E. coli and Candida species in a mixed-species environment mutually modulate biofilm development, both quantitatively and qualitatively, and that E. coli LPS appears to be a key component in mediating these outcomes.

Human serum promotes Candida albicans biofilm growth and virulence gene expression on silicone biomaterial.

Objectives Systemic candidal infections are a common problem in hospitalized patients due to central venous catheters fabricated using silicone biomaterial (SB). We therefore evaluated the effect of human serum on C. albicans biofilm morphology, growth, and the expression of virulence-related genes on SB in vitro. Methods We cultivated C. albicans SC5314 (wild-type strain, WT) and its derivative HLC54 (hyphal mutant, HM) for 48 h in various conditions, including the presence or absence of SB discs, and human serum. The growth of planktonic and biofilm cells of both strains was monitored at three time points by a tetrazolium salt reduction assay and by scanning electron microscopy. We also analyzed by RT-PCR its expression of the virulence-related genes ALS3, HWP1, EAP1, ECE1, SAP1 - SAP10, PLB1, PLB2, PLC and PLD. Results At each time point, planktonic cells of WT strain cultured in yeast nitrogen base displayed a much higher expression of EAP1 and HWP1, and a moderately higher ALS3 expression, than HM cells. In planktonic cells, expression of the ten SAP genes was higher in the WT strain initially, but were highly expressed in the HM strain by 48 h. Biofilm growth of both strains on SB was promoted in the presence of human serum than in its absence. Significant upregulation of ALS3, HWP1, EAP1, ECE1, SAP1, SAP4, SAP6 - SAP10, PLB1, PLB2 and PLC was observed for WT biofilms grown on serum-treated SB discs for at least one time point, compared with biofilms on serum-free SB discs. Conclusions Human serum stimulates C. albicans biofilm growth on SB discs and upregulates the expression of virulence genes, particularly adhesion genes ALS3 and HWP1, and hydrolase-encoding genes SAP, PLB1 and PLB2. This response is likely to promote the colonization of this versatile pathogen within the human host.

Synergistic activity of lysozyme and antifungal agents against Candida albicans biofilms on denture acrylic surfaces.

UNLABELLED Denture related oral candidiasis is a recalcitrant fungal infection not easily resolved by topical antifungals. The antimycotic protein lysozyme, in saliva is an important host defense mechanism although its activity against Candida biofilms on denture acrylic has not been evaluated. OBJECTIVES (i) To establish a clinically relevant denture acrylic assay model to develop standardized Candida albicans biofilms, and (ii) assess the inhibitory effects of lysozyme alone and, the latter combined with antifungals (nystatin, amphotericin B, ketoconazole and 5-fluorocytosine) on sessile Candida cells and, finally (iii) to visualize the accompanying ultrastructural changes. DESIGN The rotating-disc biofilm reactor was used to develop standardized 48 h Candida biofilms on acrylic discs in YNB/100 mM glucose medium and the biofilm metabolic activity was monitored using a tetrazolium reduction assay. RESULTS The biofilm metabolic activity was similar in 18 identical denture acrylic discs (p<0.05) thus validating the rotating-disc biofilm model. Very low concentrations of lysozyme (6.25 microg/ml) significantly (p<0.01) inhibited Candida biofilm formation indicating that lysozyme may likely regulate intra-oral Candida biofilm development. Although 100 microg/ml lysozyme killed 45% of sessile Candida cells, further increasing its concentration (up to 240 microg/ml) had no such effect. Nystatin, amphotericin B, and ketoconazole in association with 100 microg/ml lysozyme exhibited effective synergistic killing of biofilm Candida in comparison to drug-free controls. Scanning electron and confocal scanning laser microscopy analysis confirmed the latter trends. CONCLUSION Our results indicate that agents found in biological fluids such as lysozyme could be a safe adjunct to antifungals in future treatment strategies for recalcitrant candidal infections.

Phenotypic evaluation of the effect of anaerobiosis on some virulence attributes of Candida albicans

The current assumption that Candida albicans is a facultatively anaerobic organism has been widely accepted since its recovery from anoxic sites became common. However, the link between anaerobiosis and virulence remains uncertain. This study investigated the differential cell-surface hydrophobicity (CSH) using a hydrocarbon/water partition technique and analysed the differential secretion rates of secretory aspartyl proteases (Saps), esterase, chondroitinase and haemolysins of C. albicans strains recovered from periodontal pockets and non-periodontium-related intra-oral sites. For the enzymic tests, all strains from both sets were grown under aerobic and anaerobic conditions and the harvested cells were inoculated onto suitable normal or pre-reduced culture media in the presence or absence of molecular oxygen, respectively. The results showed that no variations were perceptible for CSH and chondroitinase (P>0.05). The secretion rates of esterase and haemolysins strongly decreased in an anoxic environment (P<0.0001). However, a consistent increment (P<0.0001) in Sap secretion was detected when cultures were grown under anaerobic conditions. Based on these results, it is suggested that the oxygen concentration in the atmosphere surrounding cells exerts a variable influence on the virulence attributes of C. albicans.