[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Mixed Candida-bacterial biofilms in urinary catheters are common in hospitalized patients. (i) The aims of this study were to evaluate, quantitatively and qualitatively, the in vitro development of mono- and dual-species biofilms (MSBs and DSBs) of Candida albicans and two enteric gram-negative bacilli (EGNB; Pseudomonas aeruginosa or Escherichia coli) on Foley catheter (FC) discs, (ii) to determine the biofilm growth in tryptic soy broth or glucose supplemented artificial urine (AU) and (iii) to assess the inhibitory effects of EGNB and their lipopolysaccharides (LPS) on Candida biofilm growth. The growth of MSBs and DSBs on FC discs was monitored by cell counts and SEM. The metabolic activity of LPS-treated Candida biofilms was determined by the XTT reduction assay. Candida albicans and EGNB demonstrated significant inter- and intra-species differences in biofilm growth on FC discs (p < 0.01). Pseudomonas aeruginosa suppressed Candida albicans significantly (p < 0.001) in DSBs. Compared with MSBs, DSB of EGNB in glucose supplemented AU demonstrated robust growth. Escherichia coli and its LPS, significantly suppressed Candida biofilm growth, compared with Pseudomonas aeruginosa and its LPS (p < 0.001). Candida albicans and EGNB colonization in FC is significantly increased in AU with glucose, and variably modified by Escherichia coli, Pseudomonas aeruginosa and their corresponding LPS.
[Show abstract][Hide abstract] ABSTRACT: Virulence associated with fluconazole (FL) resistance in Candida glabrata is a global problem and has not been well characterized at the proteome level. In this study, a stable FL-resistant (MIC >256 μg ml-1) strain of C. glabrata was generated on agar containing FL. Eight phenotypic mutants were characterized by contour-clamped homogeneous electrophoretic field analysis and two-dimensional PAGE. The secondary derivatives of C. glabrata yielded four distinct genotypes with varying chromosomal profiles. Proteomic analysis performed by tandem mass spectrometry for two of the mutants, CGL2 and CGS3, demonstrated a total of 25 differentially regulated proteins of which 13 were upregulated and 12 were downregulated or were similar compared with the parental isolate. The mRNA transcript levels of significantly (P<0.001) upregulated genes were determined by quantitative RT-PCR analysis, and their physiological relevance in terms of phenotypic expression of virulence attributes was verified by conventional laboratory methodologies. The data showed that the FL resistance (MIC >256 μg ml-1) of CGS3 was associated with significantly upregulated (P<0.001) mRNA transcript levels of several genes - ERG11, CDR1, CDR2, MFS, MTI, TPR, VPS and EFT2 - in addition to a number of other potentially virulent genes expressed differentially at a lower level. The results demonstrated accentuated phenotypic expression of bud formation of yeast and metallothionein production associated with FL resistance in C. glabrata, which may help the fungus to colonize the host.
Full-text · Article · Jan 2012 · Journal of Medical Microbiology
[Show abstract][Hide abstract] ABSTRACT: Indwelling catheters represent an important risk factor associated with mixed-candida-bacterial infections. Objectives: a) to evaluate, quantitatively and qualitatively, the development of dual-species biofilms (DSBs) of Candida and Pseudomonas aeruginosa or Escherichia coli on catheters and compare with their mono-species biofilm (MSB) counterpart, b) determine the effect of glucose on biofilm growth and c) assess the inhibitory effects of bacterial lipopolysaccharides (LPS) on candidal growth. Methods: Biofilm growth of the microorganisms on catheters was monitored in tryptic-soy-broth medium during varying time intervals and growth conditions using a viable cell count method and confirmed by scanning electron microscopy (SEM) and confocal scanning laser microscopy (SCLM). Results: Candida and the bacterial species demonstrated significant inter- and intra- species differences (p< 0.01) in biofilm growth on catheter coupons. Candida species biofilms also demonstrated variable ultrastructural differences on catheter coupons. DSBs with P. aeruginosa significantly suppressed (P <0.001) the biofilm of C. albicans. In contrast, C. albicans biofilm growth was not inhibited in mixed biofilms of E. coli and Candida, and a growth stimulatory effect was seen. These results were also confirmed by microscopy. In glucose supplemented medium C. albicans did not demonstrate a significant difference in MSB or DSB activity whereas P. aeruginosa and E. coli showed increased biofilm growth in both MSB and DSBs compared with controls. Further, LPS of E. coli was more effective in inhibiting C. albicans biofilm growth than P. aeruginosa, LPS. Conclusion: Various bacterial species appear to modulate catheter colonization by C. albicans.
[Show abstract][Hide abstract] ABSTRACT: Background: Resistance to the azole antifungal fluconazole in C. glabrata is a global problem in compromised patient populations. Objectives: To evaluate quantitatively the specific protein profiles regulated by C. glabrata in response to acquired fluconazole resistance, and to validate the possible role of these proteins in fungal virulence. Methods: A genotypically stable off shoot of C. glabrata ATCC2001 (CG) highly resistant to fluconazole (Cg) was obtained by sequential drug exposure. On subculture to RPMI/FL agar Cg produced a large number of petite yeast colonies yielding different chromosomal profiles (by CHEF analysis). Distinct differences in proteomic expression between a stable petite isolate (CgL) and the original C. glabrata strain (CG) were identified by mass spectrometry following high resolution two dimensional gel electrophoresis (2DE). Proteins implicated in virulence attributes were spotted and the expression of their mRNA transcript levels were also measured. Moreover we demonstrate the physiological role of TPR and MTI gene expression by conventional methodology. Results: Eight petite colonies yielded different patterns of chromosomal profiles (some with minor and some with major chromosome size polymorphisms). Three small colonies and two of each of the large colonies demonstrated similar chromosomal patterns. Therefore two distinct genotypes were obtained for each of the colony groups. Proteomic analysis revealed distinct and reproducible differences in the protein profiles of one stable revertant and the original C. glabrata strain. Furthermore, the relative expression level of the following genes were affected; i.e., ERG11, MTI, MTIIa, TPR, EF2, MFS and VPS . Apart from the latter three, the mRNA transcript expression levels for the remaining five genes were significantly (p<0.001) upregulated. Subsequent phenotypic studies confirmed the higher expression of TPR and MTI . Conclusion: Further proteomic studies should prove an invaluable tool in understanding the biological complexicities associated with fluconazole resistance of Candida glabrata infections.
[Show abstract][Hide abstract] ABSTRACT: Aim:
Candida glabrata is a major pathogen in humans known to be intrinsically resistant to fluconazole. However, genotypic, phenotypic, and proteomic changes associated with reduced susceptibility to fluconazole are not properly understood. The aim of this study was to observe specific phenotypic, chromosomal, and proteomic alterations in a Candida glabrata strain sequentially exposed to fluconazole.
Candida glabrata was exposed to increased concentrations of fluconazole in RPMI for 55 days. Phenotypic changes were evaluated using standard assays. Molecular/proteomic changes in C. glabrata were analyzed by contour-clamped homogeneous electric field electrophoresis, reverse transcription-polymerase chain reaction, and mass spectrometry.
Candida glabrata demonstrated increased fluconazole resistance (>256 μg/mL), with extensive cross-resistance to ketoconazole (0.38-3.0 μg), itraconazole (8 to >32 μg), and voriconazole (0.125-1.5 μg). Morphologically dissimilar colonies on RPMI/fluconazole agar demonstrated variable chromosomal profiles compared with the control isolate. Stable chromosomal changes were associated with a significantly higher (P<0.05) mRNA level of the hemolysin gene compared with the control. Phenotypic switching on CuSO4 agar was associated with variable metallothionein mRNA transcription levels. The proteome analysis of a fluconazole-resistant offshoot demonstrated a total of 98 protein spots, 25 showing a twofold upregulation.
Fluconazole exposure initiates the chance evolution of a new colonizing population with specific virulence traits.
No preview · Article · Feb 2011 · Journal of investigative and clinical dentistry
[Show abstract][Hide abstract] ABSTRACT: The post-antifungal effect (PAFE) has been shown to affect Candida pathogenicity, but there is little information on either PAFE or its association with the colonization traits of Candida glabrata. The objective of this study was to determine, in vitro, the PAFE on 14 C. glabrata isolates following exposure to amphotericin B (AMB), nystatin (NYS), ketoconazole (KETO) and 5-fluorocytosine (5FC). In addition, we evaluated the impact of PAFE on yeast adherence to buccal epithelial cells (BEC), cell-surface-hydrophobicity (CSH) and biofilm growth (BG) on denture acrylic surfaces. PAFE was induced following a 1-h exposure of yeasts to (x1-x4MIC) of AMB, NYS, KETO and 5FC in RPMI medium and, measured using automated turbidometry. The BEC adhesion, CSH and BG assays were performed by the methods of Kimura & Pearsall, Sweet et al., and Jin et al., respectively. Significant differences in PAFE (P < 0.001) were observed after exposure to AMB and NYS, but not KETO and 5FC. Following exposure to AMB, NYS, KETO and 5FC, significant inter-strain differences (P < 0.001) were observed in percentage terms in adhesion (39.0%, 43.48%, 38.28%, 35.07%) and biofilm growth (42.86%, 39.86%, 42.81%, 36.38%), respectively. Short exposure of C. glabrata to sub-cidal concentrations of antifungals modulates yeast growth and also affects some of their colonization traits.
Full-text · Article · Aug 2010 · Medical mycology: official publication of the International Society for Human and Animal Mycology
[Show abstract][Hide abstract] ABSTRACT: Candida glabrata is a major pathogen in compromised patient populations due to its increased resistance to fluconazole (FL). Although the increase in fluconazole resistance is associated with changes in its genotype no parallel phenotypic or proteomic changes have been studied. Objectives: To observe specific phenotypic, chromosomal and proteomic alterations in a C. glabrata strain sequentially exposed to fluconazole. Methods: C. glabrata was subjected to serial exposure of minimum concentrations (MICs) of fluconazole in RPMI medium for a period of 55 days. Phenotypic changes (i.e., resistance to azole drugs, adhesion to BECs, phospholipase, haemolysin production, phenotypic switching) were evaluated at different time points using standard assays. The development of molecular/ proteomic changes in C. glabrata were analyzed by contour-clamped-homogeneous electrophoretic field (CHEF) electrophoresis, RT-PCR, 2-DE gel based methodology and mass spectrometric peptide finger print analysis of SDS-PAGE yeast separated protein fractions. Results: C. glabrata demonstrated increased FL resistance (8->256g/ml) accompanied with extensive cross-resistance to ketoconazole (0.38-3.0g), itraconazole (8->32g) and voriconazole (0.125-1.5g). Variable colony morphology (size), a decrease in viable yeast counts, and inconsistent phenotypic switching on CuSO4 agar was observed. Further, seven randomly selected morphologically dissimilar yeast colonies also demonstrated pronounced differences in their chromosomal profiles compared with the control isolate. Stable changes in the chromosomal profiles of two yeast colonies were associated with significantly (p<0.05) higher transcript levels of the haemolysin gene relative to the control. Proteome analysis of one stable colony resulted in a total of 98 spots with 25 upregulated and 24 downregulated proteins. Conclusion: These findings show that sequential fluconazole exposure initiates the chance evolution of a new colonizing population with specific virulence traits. (Supported by the Research Grants Council, University of Hong Kong, SAR. We are grateful for the Pfizer Laboratories for supplying Fluconazole for this study).
[Show abstract][Hide abstract] ABSTRACT: The human fungal pathogen Candida is able to form biofilms in almost all the medical devices in current use. Indeed, biofilm formation is a major virulence attribute of microorganisms and account for a majority of human infections. Therefore, understanding processes appertaining to biofilm development is an important prerequisite for devising new strategies to prevent or eradicate biofilm-related infections. In the present study we used an array of both conventional and novel analytical tools to obtain a comprehensive view of Candida biofilm development. Enumeration of colony forming units, colorimetric (XTT) assay, Scanning Electron Microscopy (SEM) and novel Confocal Laser Scanning Microscopy (CLSM) coupled with COMSTAT software analyses were utilised to evaluate growth kinetics; architecture and viability of biofilms of a reference (ATCC) and a clinical strain each of two Candida species, C. albicans and C. glabrata. Biofilm growth kinetics on a polystyrene substrate was evaluated from the initial adhesion step (1.5 h) up to 72 h. These analyses revealed substantial inter- and intra-species differences in temporal organisation of Candida biofilm architecture, spatiality and cellular viability, while reaching maturity within a period of 48 h, on a polystyrene substrate. There were substantial differences in the growth kinetics upon methodology, although general trend seemed to be the same. Detailed architectural analysis provided by COMSTAT software corroborated the SEM and CSLM views. These analyses may provide a strong foundation for down stream molecular work of fungal biofilms.
No preview · Article · Sep 2009 · Archives of oral biology
[Show abstract][Hide abstract] ABSTRACT: Candida is the most common human fungal pathogen that causes a variety of afflictions from superficial mucosal infections to deep mycoses. Biofilm formation is a major virulence factor of Candida, and more than 300 articles have been published on Candida biofilms over the past two decades. However, most of these data are on monospecies biofilms of Candida, and information on mixed-species Candida biofilms or bacteria-Candida combinations is still scarce. Yet, in nature, the yeast exist in a mixed milieu either in the oral cavity or in other habitats with a multitude of bacteria colonising mucosal surfaces within a shared community. This mini review describes the current knowledge on candidal-candidal or bacterial-candidal interactions in mixed-species biofilms. The underlying mechanisms of these interactions appear to depend on several factors relating to biofilm development, such as species and strains of organisms, nutritional factors, aerobiosis and related environmental factors. Although the fundamental nature of these interactions appears to be commensalism and antagonism, the emerging evidence based on novel molecular, proteomic and imaging tools indicates these biological mechanisms to be far more complex than hitherto recognised. Demystifying the mechanisms underlying the growth and development of mixed-species communities involving Candida will undoubtedly yield useful data for the effective management of microbial infections in general.
[Show abstract][Hide abstract] ABSTRACT: Candida glabrata infections are increasingly seen in compromised individuals. One reason for this is likely to be the exposure of the yeast to low concentrations of antifungals associated with the diluent effect of body fluids such as serum and saliva which in turn may impact on their phenotype. Objective: To determine the post antifungal effect (PAFE) of 15 C. glabrata isolates following limited exposure to minimum inhibitory concentrations (MICs) of amphotericinB (AmB), nystatin (NYS), ketoconazole (KETO) and 5-fluorocytosine (5-FC) and secondly, to evaluate its impact on yeast adherence to buccal epithelial cells (BEC), their cell surface hydrophobicity (CSH) and biofilm growth (BG) on denture acrylic surfaces. Methods: PAFE was induced in fifteen C. glabrata isolates following a 1hr exposure to (1-4MIC) of AmB, NYS, KETO and 5-FC in RPMI medium and measured using an automated turbidometric method. The virulent attributes of six yeast isolates exposed to the four drugs were assessed by standard assays. Results: Significant PAFEs (P<0.001) were observed for the majority of yeast isolates exposed to AmB and NYS, a reduced PAFE for KETO and none for 5-FC. The mean duration of the PAFEs for 4MIC of AmB, NYS, KETO and 5-FC were 16.22 5.10h, 7.90 4.40h, 3.55 1.46h and 5.81 3.81, respectively. Following exposure to the drugs (i.e., AmB, NYS, KETO and 5-FC) the mean percentage reduction of adhesion to BECs, CSH and BG on denture acrylic of C. glabrata were 39.48%, 45.22%, 39.89%, 35.79%; 0%, 7.92%, 0%, 7.08% and 43.33, 40.22, 43.22, 36.30, respectively with significant interspecies differences (P<0.001). Conclusion: These findings imply that exposure of C. glabrata to varying sub-therapeutic concentrations of antifungals modulate their growth and, impacts on their adhesion to BECs, CSH and BG on denture acrylic surfaces.
[Show abstract][Hide abstract] ABSTRACT: 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.
(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.
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.
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.
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.
No preview · Article · Dec 2008 · Archives of oral biology
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study is to compare the light and scanning electron microscopic (SEM) features of tissue invasion by three Candida species (C. albicans, C. tropicalis, and C. dubliniensis) in two different tissue culture models: rabbit tongue mucosal explants (RTME) and reconstituted human oral epithelium (RHOE). Tongue mucosal biopsies of healthy New Zealand rabbits were maintained in explant culture using a transwell system. RHOE was obtained from Skinethic Laboratory (Nice, France). RTME and RHOE were inoculated with C. albicans, C. tropicalis, and C. dubliniensis separately and incubated at 37 degrees C, 5% CO(2), and 100% humidity up to 48 h. Light microscopic and SEM examinations of uninfected (controls) and infected tissues were performed at 24 and 48 h. C. albicans produced characteristic hallmarks of pathological tissue invasion in both tissue models over a period of 48 h. Hyphae penetrated through epithelial cells and intercellular gaps latter resembling thigmotropism. SEM showed cavitations on the epithelial cell surfaces particularly pronounced at sites of hyphal invasion. Some hyphae on RTME showed several clusters of blastospores attached in regular arrangements resembling "appareil sporifere". C. tropicalis and C. dubliniensis produced few hyphae mainly on RTME but they did not penetrate either model. Our findings indicate that multiple host-fungal interactions such as cavitations, thigmotropism, and morphogenesis take place during candidal tissue invasion. RTME described here appears to be useful in investigations of such pathogenic processes of Candida active at the epithelial front.
[Show abstract][Hide abstract] ABSTRACT: Biofilm formation is a major virulence attribute of Candida pathogenicity which contributes to higher antifungal resistance. We investigated the roles of cell density and cellular aging
on the relative antifungal susceptibility of planktonic, biofilm, and biofilm-derived planktonic modes of Candida. A reference and a wild-type strain of Candida albicans were used to evaluate the MICs of caspofungin (CAS), amphotericin B (AMB), nystatin (NYT), ketoconazole (KTC), and flucytosine
(5FC). Standard, NCCLS, and European Committee on Antibiotic Susceptibility Testing methods were used for planktonic MIC determination.
Candida biofilms were then developed on polystyrene wells, and MICs were determined with a standard 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide assay. Subsequently, antifungal susceptibility testing was performed for greater inoculum concentrations
and 24- and 48-h-old cultures of planktonic Candida. Furthermore, Candida biofilm-derived planktonic cells (BDPC) were also subjected to antifungal susceptibility testing. The MICs for both C. albicans strains in the planktonic mode were low, although on increasing the inoculum concentration (up to 1 × 108 cells/ml), a variable MIC was noted. On the contrary, for Candida biofilms, the MICs of antifungals were 15- to >1,000-fold higher. Interestingly, the MICs for BDPC were lower and were similar
to those for planktonic-mode cells, particularly those of CAS and AMB. Our data indicate that higher antifungal resistance
of Candida biofilms is an intrinsic feature possibly related to the biofilm architecture rather than cellular density or cellular aging.
[Show abstract][Hide abstract] ABSTRACT: The in vitro lysozyme susceptibility of three oral isolates of Candida albicans cultured in carbohydrate-supplemented media was studied. Lysozyme was shown to have a dose- and time-dependent killing effect on C. albicans isolates. Fungieidal activity persisted to varying degrees when yeast isolates were cultured in a variety of carbohydrates (glucose, galascrose. sucrose, maltose. xylitol and laelose) before exposure to 20 μg/ml lysozyme. Sucrose and galactose grown yeasts exhibited increased resistance to iysozyne conipared with (in decreasing order) those grown in glucose, maltose, xylnol or laelose. Further, the C albicans isolates tested demonstrated strain variations in their susceptibility to lysozyme. These results suggesl that dietary carbohydrate may play a role in modulating the yeast cell populations in the oral —– by altering the fungal susceptibility to salivary lysozyme.
No preview · Article · Dec 2007 · Oral Microbiology and Immunology
[Show abstract][Hide abstract] ABSTRACT: Biofilms contribute to the pathogenesis of oral candidiasis, some 15% of which may be due to dual Candida species. Despite extensive studies on monospecies biofilms (MSB) on denture acrylic surfaces, few have investigated the characteristics of dual species Candida biofilms (DSB).
To examine interactions of DSB of Candida albicans and Candida krusei on denture acrylic surfaces.
Two isolates each of C. albicans (Ca) and C. krusei (Ck), with high (Ca(h), Ck(h)) and low (Ca(l), Ck(l)) biofilm-forming ability were used. The biofilms were developed on acrylic surfaces aerobically at 37 degrees C in yeast nitrogen base (YNB) medium, and growth quantified by colony-forming unit (CFU) assay. We determined: (i) the population profiles of DSB comprising each pair of Candida species, of a total of four combination pairs, after 12 h, (ii) the effect of a constant concentration of Ca(h) (10(7)cells/ml) on varying concentrations of Ck(h) (10(3)-10(7)cells/ml) on DSB development and (iii) the effect of saliva on the growth of DSB.
(i) DSB exhibited a lower cell population after 9 or 12 h in comparison to MSB (P<0.05), (ii) C. albicans (10(7)cells/ml) co-cultured with varying initial concentrations of C. krusei was inhibited at high concentrations of the latter (10(6)-10(7)cells/ml) (P<0.05) and (iii) only the MSB development of C. krusei was affected by saliva (P<0.05).
Our data suggest that the competitive interactions of fungal species are likely to be important in biofilm formation on acrylic surfaces and human saliva may further modulate this process.
No preview · Article · Dec 2007 · Archives of Oral Biology
[Show abstract][Hide abstract] ABSTRACT: An understanding of biofilm behavior of Candida species under different environmental conditions is key to the development of effective preventive measures for candidal infections. Hence in this study we assessed the impact of the environmental milieu on Candida biofilm formation using polystyrene, flat-bottomed 96-well microtiter plates. A total of 20, comprising 10 clinical isolates each of Candida albicans and, non-albicans species of Candida were compared for their biofilm forming ability both under aerobic and anaerobic conditions, and static and dynamic conditions. XTT reduction assay was used to quantify the sessile growth. Biofilm formation of all 10 C. albicans isolates differed significantly between dynamic and static states under both atmospheric conditions (P<0.05). For non-albicans Candida species, a significant difference in biofilm growth between dynamic and static states was noted only when incubated aerobically (P<0.05), and no significant difference in biofilm formation was noted between aerobic and anaerobic conditions. Scanning electron microscopy revealed that C. albicans produced a compact multilayered biofilm embedded in noticeably higher quantity of extracellular polymeric matrix in aerobic/dynamic conditions compared with anaerobic/static conditions. Our data indicate that biofilm formation of C. albicans and non-albicans Candida species is modulated by hydrodynamic conditions and ambient oxygen gradients. However, further work is required to fully elucidate how Candida biofilms persist within the oral milieu under such challenging ecological pressures.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Oral candidiasis is a common problem in compromised patients. Although several non-albicans Candida species have emerged as pathogens the majority of candidal infections are caused by Candida albicans. Morphogenesis from the blastospore to filamentous phase, and production of secretory aspartyl proteinases (SAP) are two major virulence attributes of these opportunistic yeast. Histopathology of oral candidiasis is characterized by fungal invasion of the superficial epithelium although the invasive potentials of different Candida species vary. Computerized image analysis systems (IAS) utilizing immunohistochemistry have been successfully employed for quantification of such histopathological features. The purpose of this study was to evaluate quantitatively the in vitro invasive potential of C. albicans and its hyphal and SAP mutants, and five other non-albicans Candida species using a computerized IAS.
In vitro human oral candidiasis was produced using five wild type and one reference C. albicans isolates, hyphal and SAP mutants of C. albicans SC 5314, and one wild type and one reference isolate each of C. tropicalis, C. dubliniensis, C. glabrata, C. parapsilosis and C. krusei in a reconstituted human oral epithelium (RHOE) model. The infected tissues were examined histologically at 12, 24 and 48 h. Invading fungal elements were visualized by periodic acid-Schiff (PAS) staining and quantitatively evaluated as a percentage of total tissue invasive area, using a computerized IAS.
All C. albicans isolates including hyphal mutant cph1/cph1 and SAP mutants; sap 1-3, sap 4-6 produced hyphae and differentially (P < 0.05) invaded the tissue over 48 h. The invasive potential of hyphal mutant cph1/cph1 and SAP mutants (sap 1-3, sap 4-6) were similar to the parent wild-type isolate at 12 h although after 24 h their invasion was dissimilar (P < 0.05). Non-albicans Candida species and hyphal mutants; efg1/efg1, efg1/efg1 cph1/cph1 were all non-invasive.
RHOE model in combination with computerized image analysis permits for the first time, the assessment of invasive potential of Candida species in a quantitative manner. The differential tissue invasive patterns of various C. albicans isolates, their mutants and other Candida species are also described.
No preview · Article · Oct 2006 · Journal of Oral Pathology and Medicine
[Show abstract][Hide abstract] ABSTRACT: To study molecular profiles of oral Candida tropicalis isolates from five different geographic locales to determine the molecular diversity, clonality and evolutionary trends of this opportunistic pathogen.
A total of 36 strains from five countries (China, Canada, Scotland, Japan and Tanzania) were genotyped by PCR fingerprinting with 11 separate primers. Of these, primers RSG9, RSG8, T3B and RSD12 generated complex fingerprinting patterns.
Three significantly dissimilar profiles were derived from the primer T3B and particularly focused on tDNA suggested the prevalence of genetic subtypes within the species. Comparison of tDNA and rDNA (RSD12) fingerprints of C. tropicalis suggested that rDNA is much more heterogeneous than the relatively distinct tDNA. Further analysis of similarity coefficient (SAB) of gel profiles derived from computer-generated dendrograms indicated some degree of similarity in isolates from five-disparate geographic locales as well as the presence of unique isotypes in each region.
This study demonstrates the evolutionary divergence of distinct genetic subgroups within Candida tropicalis .
No preview · Article · Aug 2006 · Indian Journal of Medical Microbiology