Acid proteinase,phospholipase, and biofilm production of <Emphasis Type="Italic">Candida</Emphasis> species isolated from blood cultures

Three virulence factors comprising proteinase, phospholipase, and biofilm among 68 Candida albicans and 31 non-albicans Candida strains (11 C. tropicalis, 8 C. parapsilosis, 6 C. glabrata, 4 C. guillermondii, 2 C. krusei) isolated from blood cultures were analyzed. In total, 61 (89.7%) C. albicans strains were detected as proteinase positive whereas eight (25.8%) non-albicans Candida strains were proteinase positive (P < 0.05). Phospholipase production was detected in 41 (60.3%) C. albicans strains. All non-albicans Candida strains were phospholipase negative. Biofilm production was determined by both visual and spectrophotometric methods. Eight (11.8%) of C. albicans strains and 13 (41.93%) of 31 non-albicans Candida strains were biofilm positive with two of the methods (P < 0.05). According to our results, we may suggest that detection of hydrolytic enzyme and biofilm production abilities of the Candida isolates in clinical mycology laboratories may warn the clinican for a possible hematogenous infection.     

Distribution and phospholipase activity of Candida species in different denture stomatitis types

The aim of this study was to evaluate the correlation between frequency and phospholipase activity of Candida species and denture stomatitis according to Newton’s classification. Seventy-five complete denture wearers were evaluated for the presence of yeasts on the palatal mucosa by culture method. In addition, the number of yeast isolates producing phospholipase and amount of this enzyme were determined using egg yolk agar plate method. According to Newton’s classification, 25 denture wearers were with healthy palatal mucosa while 50 were with any types of denture stomatitis. The frequency of yeasts was linked to whether subjects had Type II or Type III, but not Type I denture stomatitis. Candida albicans was the most frequently isolated species in denture wearers with and without clinical signs of denture stomatitis and it was the only species produced phospholipase. Although the amount of phospholipase produced by the C. albicans isolates from denture wearers in control and Type II and III DS groups was not significantly different, there was statistically significant difference in the number of C. albicans isolates producing phospholipase between patients with and without clinical signs of DS.     

Effect of phenotypic switching on expression of virulence factors by Candida albicans causing candidiasis in diabetic patients

A total of 110 strains belonging to seven species of Candida were isolated from various forms of candidiasis in diabetic patients. They were Candida albicans 53 (47%), Candida tropicalis 36 (33%), Candida glabrata 9 (8%), Candida parapsilosis 4 (4%), Candida guilliermondii 2 (2%), Candida krusei 5 (5%) and Candida kefyr 1 (1%). All 53 strains of C. albicans isolated were observed to express virulence factors such as cell surface hydrophobicity (CSH), adherence to human buccal epithelial cell (BEC) and proteinase activity (100%), while phospholipase activity was observed in 52 (98%). Phenotypic switching and its influence on the pathogenicity of C. albicans were studied. Two C. albicans strains isolated from oral and vaginal thrush, respectively, in diabetic individuals, and the control strain C. albicans NCPF 3153A were induced to undergo phenotypic switching by exposure to UV light and the degree of expression of virulence factors by the different morphological forms was determined. Three different morphological forms of C. albicans were obtained, namely Star (S), Wrinkled (W) and Ring (R) types from the original Smooth (O) variety. It was found that proteinase activity was greatest with the W type followed by the R type then the O type. The S type produced the least proteinase. The phospholipase activity was greatest with O type followed by R type. The W and S types produced the least phospholipase. Expression of CSH and adherence was greatest in the O type followed by the R and then the W type and finally the S type. Differential expression of virulence factors occurs with different phenotypic forms of C. albicans and this may provide a particular morphological type with a distinct advantage over other types in causing candidiasis.     

Determining <Emphasis Type="Italic">Candida</Emphasis> spp. Incidence in Denture Wearers

The aim of this study was to determine Candida spp. incidence in the oral cavity of denture wearers and characterize predisposing factors in denture-related stomatitis (DRS). Three groups of denture wearers and a control group were evaluated for DRS according to Newton’s classification. The amount of yeast in saliva and the presence of yeast on mucosal surfaces were determined by phenotyping methods, and the impact of some risk factors on candidal carriage was evaluated. The development of DRS is most common in complete prosthesis users. When the count of yeast in saliva is ≥400 cfu/ml, the frequency of DRS is increased. In individuals who develop DRS, the most frequently encountered species that was identified as C. albicans. Prosthetic hygiene was related to the intensity of candidal growth and the development of DRS. C. albicans live as saprophyte in the oral cavity. But, it is capable of causing infection if there are predisposing conditions related to the host. Usage of removable prosthesis may cause these microorganisms to gain pathogenicity.     

Adhesive Properties and Hydrolytic Enzymes of Oral <Emphasis Type="Italic">Candida albicans</Emphasis> Strains

Several virulence factors in Candida albicans strains such as production of hydrolytic enzymes and biofilm formation on surfaces and cells can contribute to their pathogenicity. For this, control of this opportunistic yeast is one of the factors reducing the nosocomial infection. The aim of this study was to investigate biofilm formation on polystyrene and polymethylmethacrylate and the production of hydrolytic enzymes in Candida albicans strains isolated from the oral cavity of patients suffering from denture stomatitis. All strains were identified by macroscopic, microscopic analysis and the ID 32 C system. Our results showed that 50% of the total strains produced phospholipase. Furthermore, protease activity was detected in seven (35%) strains. All Candida albicans strains were beta haemolytic. All C. albicans strains adhered to polystyrene 96-well microtiter plate at different degrees, and the metabolic activity of C. albicans biofilm formed on polymethylmethacrylate did not differ between tested strains. The atomic force micrographs demonstrated that biofilm of Candida albicans strains was organized in small colonies with budding cells.     

Genotypic analysis of Candida albicans isolates obtained from removable prosthesis wearers

Aims: To assess of the genotypic diversity of Candida albicans isolated from removable prosthesis wearers, with and without denture‐related stomatitis (DRS). The occurrence of different genotypes in pathological and control cases was investigated. Methods and Results: One hundred and sixty‐four isolates of C. albicans obtained from different oral cavity locations were compared by randomly amplified polymorphic DNA (RAPD). The coherence of this analysis was confirmed by genotyping a selected group of isolates with pulsed field gel electrophoresis (PFGE). Among the 164 isolates, 150 were grouped into seven groups on the basis of their RAPD patterns. Three of these groups (comprising 54 isolates) had significant (α < 0·10) predominance of clinical or control cases. For the other isolates, no significant differences were observed between control and DRS cases. Occasionally, more than one genotype was found in the same person. These findings were sustained by PFGE analysis. No relevant associations between the genotypic patterns and pathology level were found. Conclusions: This study evidenced that C. albicans with similar genotypes may be found in individuals with DRS and in control cases. Significance and Impact of the Study: This conclusion hints the involvement of other aetiological factors that alone or in association with C. albicans may trigger the emergence of DRS.     

Epidemiology and molecular typing of Candida isolates from burn patients

This study, spread over a span of 2 years describes Candida infections in burn patients of an Indian hospital. A total of 220 burn patients were monitored and Candida could be isolated from 138 patients. A total of 228 different Candida species were obtained from various body locations of these patients. Species identification revealed that Candida albicans was the most predominant (45) followed by Candida tropicalis(33), Candida glabrata (13.5), C. parapsilosis (4), C. krusei (2.75) and C. kefyr (1.75). DNA fingerprinting of all C. albicans isolates was done by using CARE-2 probe. Fingerprinting analyses of all the C. albicans strains revealed that strains collected from different patients were different. It is noteworthy that patients with disseminated candidiasis had a similar, but unique strain isolated from all body locations, suggesting a possibility that commensal isolates might be turning pathogenic. Taken together, this is probably the first ever detailed survey of Candidainfections in burn patients in India and is expected to lead to better clinical management of this group of patients.     

Candida albicans and Candida tropicalis in oral candidosis: quantitative analysis, exoenzyme activity, and antifungal drug sensitivity

Candida albicans and C. tropicalis obtained from whole saliva of patients presenting signs of oral candidosis were assayed for quantification of colony forming units, exoenzyme activity (phospholipase and proteinase) and antifungal drug sensitivity (amphotericin B, fluconazole and itraconazole) by the reference method of the Clinical and Laboratory Standards Institute. The number of colony forming units per milliliter varied according to the Candida species involved and whether a single or mixed infection was present. Proteinase activity was observed in both C. albicans and C. tropicalis, but phospholipase activity was noted only in C. albicans. In vitro resistance to antifungals was verified in both species, but C. tropicalis appears to be more resistant to the tested antifungals than C. albicans.     

Phospholipase and Proteinase Activities of Clinical Isolates of Candida from Immunocompromised Patients

Sixty-one isolates of Candida recovered from HIV seropositive and cancer patients were studied for elaboration of putative virulence determinants – phospholipase (PL) and secreted aspartyl proteinase (Sap). Forty two (68.85%) isolates examined were PL producers and 51 (83.6%) were positive for Sap. 57.37% (35/61) isolates produced both enzymes. Enzymatic activity was more pronounced in Candida albicans with 100% PL and 94.1% Sap activity. In contrast, non-C. albicans species demonstrated only 29.6% PL and 70.3% Sap activity, indicating interplay of other virulence determinants in these yeasts in colonization and disease.     

Biomaterial-Associated Infection with Candida albicans in Mice

Candida yeasts are frequently isolated from patients with continuous ambulatory peritoneal dialysis peritonitis or other biomaterial-associated infections. The mouse model of candidal peritonitis was used to study the interaction of Candida cells with end-point attached heparinized polyethylene (H-PE) and with polymorphonuclear leukocytes (PMNs) or macrophages (Mφ). Two Candida strains differing in cell surface hydrophobicity and in expression of fibronectin (Fn) binding were used for the study. Cells of both Candida strains adhered at higher numbers to H-PE surfaces preadsorbed with Fn or with human dialysis fluid (HDF) than to non-modified H-PE, supporting a role of Fn in mediating adhesion. C. albicans 4016 cells expressing low hydrophobicity and low binding of soluble Fn demonstrated stronger adhesion to PMNs than the more hydrophobic C. albicans 3248 yeasts, which express high binding of soluble Fn. However, C. albicans 4016 cells were more resistant to phagocytic killing and were hardly eradicated in intraperitoneally infected mice. The animals depleted in PMNs by treatment with CY were neither able to eradicate C. albicans 3248 (rapidly eliminated by normal mice) nor C. albicans 4016 yeasts (with a tendency to persist in the tissues of normal mice).     

Correlation between factors associated with the removable partial dentures use and Candida spp. in saliva

doi: 10.1111/j.1741‐2358.2010.00390.x
Correlation between factors associated with the removable partial dentures use and Candida spp. in saliva Objectives: To correlate the presence and number of Candida spp. in the saliva of wearers of removable partial dentures retained with precision attachments with the proportion of metal/acrylic resin present in the dentures. Methods: Saliva samples from 40 removable partial denture wearers (test) and one paired sample of individuals, non‐wearers of any type of removable denture (control) were collected, seeded, and the colony forming units of Candida counted and identified. The metal/acrylic resin proportion of each denture was quantified, using silicone plates pressed over each denture. Results: Candida spp. was found in the saliva of 80% of the individuals in the test group and 65% of the control, with C. albicans being the most prevalent species. The test group presented with the highest number of colony forming units of Candida per ml of saliva, and there was weak correlation between this number and the metal and resin area of the denture (Pearson’s coefficient of correlation). Greater prevalence and a higher number of colony forming units of Candida per ml of saliva occurred in removable partial denture wearers (p = 0.04) with a weak positive correlation between the metal and resin area and the number of colony forming units of Candida per ml of saliva. However, this correlation was more significant for the area of resin.     

Impact of oxidative and osmotic stresses on Candida albicans biofilm formation

Candida albicans possesses an ability to grow under different host-driven stress conditions by developing robust protective mechanisms. In this investigation the focus was on the impact of osmotic (2M NaCl) and oxidative (5 mM H₂O₂) stress conditions during C. albicans biofilm formation. Oxidative stress enhanced extracellular DNA secretion into the biofilm matrix, increased the chitin level, and reduced virulence factors, namely phospholipase and proteinase activity, while osmotic stress mainly increased extracellular proteinase and decreased phospholipase activity. Fourier transform infrared and nuclear magnetic resonance spectroscopy analysis of mannan isolated from the C. albicans biofilm cell wall revealed a decrease in mannan content and reduced β-linked mannose moieties under stress conditions. The results demonstrate that C. albicans adapts to oxidative and osmotic stress conditions by inducing biofilm formation with a rich exopolymeric matrix, modulating virulence factors as well as the cell wall composition for its survival in different host niches.     

Candida albicans strain-dependent modulation of pro-inflammatory cytokine release by in vitro oral and vaginal mucosal models

Candida albicans is a commensal organism at several sites and is a versatile, opportunistic pathogen. The underlying factors of pathogen and host associated with commensalism and pathogenicity in C. albicans are complex and their importance is largely unknown. We aimed to study the responses of oral epithelial (OEM) and vaginal epithelial models (VEM) to infection by oral and vaginal C. albicans strains to obtain evidence of inter-strain differences in pathogenicity and of site-specificity. Following inoculation of models, proinflammatory cytokines IL-1α, IL-1β, IL-6, IL-8 and prostaglandin E2 (PGE2) release were monitored and histological staining undertaken. Striking differences in strain behaviour and epithelial responses were observed. IL-1α, IL-1β and IL-8 release were significantly increased from the OEM in response to denture stomatitis strain NCYC 1467. Increased IL-8 release also followed infection of the OEM with both vaginal strains. Overall the VEM was relatively unresponsive to infection with either oral or vaginal strains under these conditions. Adherence and hyphal development were observed for all strains on both models although extensive, uniform tissue penetration was seen only with stomatitis strain NCYC 1467 on the OEM. Candidal strains were assayed for phospholipase (PL) and secreted aspartyl proteinase (SAP) activities where phospholipase (PL) activity was highest for strain NCYC 1467 although highest SAP activity was observed for vaginal strain NCPF 8112 in this assay. This is the first study to concurrently investigate cytokine production from oral and epithelial models using candidal strains originating from these respective mucosal sites from healthy and disease states. These data demonstrate significant differences in inflammatory responses of host epithelia to individual C. albicans strains.     

Virulence Factors and Antifungal Susceptibility of Candida albicans Isolates from Oral Candidosis Patients and Control Individuals

Sixty isolates of Candida albicans, 30 obtained from the oral cavity of denture wearers presenting signs of candidosis and 30 obtained from the oral cavity of denture wearers with normal palatal mucosa were assayed for phospholipase and proteinase production, as well as for adherence to buccal epithelial cells. Likewise, susceptibility of the isolates to antifungals was determined by the NCCLS reference method and the E-test method. Proteinase activity was increased among the strains obtained from oral candidosis patients. In contrast, no significant differences between the two groups of isolates were observed in their adherence ability in vitro, in phospholipase production, and susceptibility to antifungal drugs.     

Identification of Candida isolated from the cutaneous candidiasis by the combined use of confirmatory medium and slide agglutination with monofactorial antibodies

Forty strains ofCandida and one ofTorulopsis were isolated from patients with cutaneous candidiasis. The isolates comprised 29 strains ofC. albicans, 7 strains ofC. tropicalis, 2 strains ofC. guilliermondii, and one each ofC. parakrusei, C. lipolytica, andT. famata were identified by the ordinary method. Besides the common pathogenC. albicans, a few other species ofCandida may be etiologic organisms of cutaneous candidiasis. These strains were re-examined by combined use of sucrose agar slants and slide agglutination tests with IgG monofactorial antibodies as a rapid identification method, especially for determining serotypes ofC. albicans. The new method was useful and reliable for rapid identification ofC. albicans and related species. All strains ofC. albicans isolated from skin lesions proved to be standard serotypes ofC. albicans.

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Zusammenfassung Vierzig Stämme vonCandida und eins vonTorulopsis wurden aus Kranken mit kutanen Candidamykosen isoliert. Neunundzwanzig Stämme vonC. albicans, 7 vonC. tropicalis, 2 vonC. guilliermondii, und je einer vonC. parakrusei, C. lipolytica undT. famata wurden mit dem ordinären Methode identifiziert. Außer dem gewohnlichen Erreger,C. albicans, konnten auch ein Paar andere Spezies vonCandida als den Erreger betrachtet werden. Sechsunddreißig Stämme vonC. albicans undC. tropicalis wurden mit der von uns verbesserten kombinierten serologischen und biologischen Methode untersucht, besonders um den Serotypus vonC. albicans festzusetzen. Die neue Methode war gut und zuverlässig als die rapide Identification vonC. albicans und verwandten Spezies. Alle aus der Hautläsion isoliertenC. albicans waren der in Japan allgemeine Serotypus vonC. albicans.

     

Experiments on in vivo biofilm formation and in vitro adhesion of Candida species on polysiloxane liners

doi:10.1111/j.1741‐2358.2009.00329.x
Experiments on in vivo biofilm formation and in vitro adhesion of Candida species on polysiloxane liners Objectives: Microorganisms may colonise polysiloxane soft liners leading to bio‐deterioration. The aim of this study was to investigate in vitro adhesion and in vivo biofilm formation of Candida species on polysiloxane surfaces. Methods: The materials used in this study were Molloplast B, GC Reline soft, Mollosil Plus, Silagum Comfort and Palapress Vario. The in vitro retention of clinical isolates of Candida albicans to the relining and denture‐base materials by microscopic (scanning electron microscopy, SEM), conventional culturing methods and antimicrobial properties of these materials were studied. Candida found on materials and mucosa following long‐term use were identified and quantified, and biofilms covering the surfaces were investigated by SEM. Results: There was a significant decrease in the number of cells attached in vitro to saliva‐coated surfaces compared with non‐treated surfaces. An oral Candida carriage of 78% was found. Candida albicans, C. glabrata, C. intermedia and C. tropicalis were identified. In vivo biofilm formation on the liners appeared as massive colonisation by microorganisms. Conclusions: The results of the in vitro experiments suggest that salivary film influences early colonisation of different C. albicans strains. The film layer also minimises the differences among different strains. The Candida carriage of these patients was similar to denture‐wearing patients without soft liners.     

Species Distribution and Virulence Factors of Candida spp. Isolated from the Oral Cavity of Kidney Transplant Recipients in Brazil

Although yeasts belonging to the genus Candida are frequently seen as commensals in the oral cavity, they possess virulence attributes that contribute for pathogenicity. The aims of the present study were to study the prevalence of Candida spp. isolated from the oral cavity of renal transplant recipients and to analyze strains virulence factors. We isolated a total of 70 Candida strains from 111 transplant recipients, and Candida albicans was the most prevalent species (82.86 %). Oral candidiasis was diagnosed in 14.4 % kidney transplant patients, while 11 isolates (15.7 %) corresponded to non-Candida albicans Candida (NCAC) species. C. albicans adhered to a higher extension than NCAC strains. Some isolates of Candida tropicalis were markedly adherent to human buccal epithelial cells and highly biofilm-forming strains. Regarding proteinase activity, Candida orthopsilosis was more proteolytic than Candida metapsilosis. Candida glabrata and Candida dubliniensis showed very low ability to form biofilm on polystyrene microtiter plates. We have demonstrated here diverse peculiarities of different Candida species regarding the ability to express virulence factors. This study will contribute for the understanding of the natural history and pathogenesis of yeasts belonging to the genus Candida in the oral cavity of patients who were submitted to kidney transplant and are under immunosuppressive therapies.     

Effects of Antifungal Agents in Sap Activity of <Emphasis Type="Italic">Candida albicans</Emphasis> Isolates

Some antifungal agents have shown to exert effects on expression of virulent factors of Candida as the production of secretory aspartyl proteinase (Sap). In this study, we sought to determine and to compare the influence of fluconazole and voriconazole in proteinase activity of this microorganism. Thirty-one isolates obtained from oral mucosa of human immunodeficiency virus positive (HIV⁺) patients were used in this study. The minimal inhibitory concentrations (MIC) of fluconazole and voriconazole were determined using the broth microdilution method with RPMI 1640 medium and with yeast carbon base–bovine serum albumin (YCB–BSA) medium. The Sap activity following by digestion of BSA as substrate was determined for four Candida albicans strains arbitrarily chosen according to susceptibility (susceptible or resistant) to fluconazole or voriconazole. Besides, the SAP1 to SAP7 genes were screened by PCR for the same isolates that were determined by the Sap activity. In vitro susceptibility testing using the two media presented similar MIC values. Increased Sap activity was observed in resistant isolates on presence of drugs, but the Sap activity by susceptible isolates to azoles showed different behavior on the presence of drug. We detected the presence of SAP1 to SAP7 genes from all susceptible or resistant C. albicans isolates. The present study provides important data about the proteinase activity and the presence of genes of SAP family in fluconazole and voriconazole susceptible or resistant C. albicans isolates.     

The effect of denture adhesives on Candida albicans growth in vitro

doi: 10.1111/j.1741‐2358.2011.00478.x
The effect of denture adhesives on Candida albicans growth in vitro Objective: Denture‐wearing favours the growth of Candida. In view of the fact that many denture wearers regularly use adhesives to enhance denture retention, stability and function, the aim of this work was to study the effect of denture adhesives on Candida albicans growth in vitro. Materials and methods: The denture adhesives tested were Corega^® cream, Kukident^® cream, Novafix^® cream, Polident^® cream, Protefix^® cream, Steradent^® cream, Aderyn^® powder, Corega^® ultra powder, Protefix^® powder and Corega^® strip. C. albicans growth curves were obtained in the presence or absence of a 1% solution of the denture adhesive diluted in Sabouraud broth. Macro‐ and microscopic morphological changes in C. albicans were analysed, as was microbial contamination of the denture adhesive. Results: Most of the denture adhesives studied induced morphological changes in C. albicans cells and colonies, but only two had any significant inhibitory effect on yeast growth. Kukident^® cream markedly inhibited C. albicans growth in a concentration‐dependent way, reducing the growth rate by 95%, whereas Corega^® cream also inhibited C. albicans growth but in a non‐concentration‐dependent way, reducing the growth rate by 37%. In addition, denture adhesives available as powders had detectable microbial contamination. Conclusion: Some commercially available denture adhesives showed microbial contamination and some had significant inhibitory effect on C. albicans growth.     

Electrophoretic Karyotypes of Candida Yeasts Recurrently Isolated from Single Patients

Candida strains were isolated repeatedly from single patients during recurrent episodes of Candida infection in a hospital, and their electrophoretic karyotypes were analyzed by pulsed-field gel electrophoresis using CHEF system. When only C. albicans (in 6 patients) or C. glabrata (in 1 patient) were recurrently isolated, their karyotypes from each patient were almost identical to one another, suggesting that they carried single type of the yeast. When multiple species were recovered from single patients (6 cases), the karyotypes of the most frequently recovered yeast species were almost identical with respect to each patient. The electrophoretic karyotype analysis has been proved to be useful for epidemiological studies because the method can tell not only the species identification but also the differences among the strains of the same species.