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.     

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.     

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.     

Effect of pepstatin A on the virulence factors of Candida albicans strains isolated from vaginal environment of patients in three different clinical conditions

The aspartate proteinase inhibitor pepstatin A was used to study a possible correlation among proteinase activity and other virulence factors of Candida albicans strains isolated from the vaginal environment of patients in three different clinical conditions: asympthomatic, vulvovaginal candidiasis (VVC) and recurrent vulvovaginal candidiasis (RVVC). The addition of 1.0 μM pepstatin A did not have any significant effect on hyphae formation, biofilm production and in the cell surface hydrofobicity of isolates in the three different clinical conditions. However, pepstatin A reduced the adherence of C. albicans to vaginal mucosa epithelial cells (53.1, 48.7 and 59.9%, respectively to isolates from asymptomatic, VVC and RVVC patients). This result suggests that the secreted aspartate proteinases (Saps) of this fungal pathogen may have auxiliary roles in cellular adhesion.     

Investigation of extracellular phospholipase and proteinase activities of Candida species isolated from individuals denture wearers and genotypic distribution of Candida albicans strains

In order to determine the relationship between the development of denture related stomatitis (DRS) and the production of phospholipase and proteinase by Candida species, 156 Candida isolates isolated from the individuals in the control group and from the individuals different denture wearers were included in this study. According to the results of the study, C. albicans strains were determined to produce high levels of phospholipase and proteinase. It was also determined that the prevalence of phospholipase and proteinase activities in C. albicans strains isolated from individuals with DRS and from the individuals without DRS was not different. In order to determine genotypic variation of 109 C. albicans strains isolated, CA-INT-L and CA-INT-R primers specific to the site of the transposable group I intron of the 25S rRNA gene (rDNA) region were used. As a result, it was considered that, there were several other virulence factors belonging to the microorganism which played a role in the development mechanisms of the infection caused by C. albicans. In addition, according to the results of microbial genotyping, it was determined that there were no C. albicans strains specifically responsible for the development of DRS.     

Evaluation of Antifungal Activity of Medicinal Plant Extracts Against Oral <Emphasis Type="Italic">Candida albicans</Emphasis> and Proteinases

Proteinases produced by Candida albicans are one kind of virulence factor expressed that contribute to adherence and invasion of host tissue. Proteinase inhibitor of human immunodeficiency virus in experimental candidiasis suggested reduction in fungal infection, and medicinal plants could be a source of alternative agent to prevent diseases. In this study, we investigated the production of proteinases by C. albicans from clinical isolates and the action of plant extracts against strains of C. albicans and its synthesized proteinases, comparing with antifungal fluconazole and amphotericin B and proteinase inhibitors pepstatin A, amprenavir, and ritonavir. The results reported here showed that these extracts have a certain kind of action and that the search for new antifungal agents could be found at the plants.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

Use of CHROMagar in the Differentiation of Common Species of <Emphasis Type="Italic">Candida</Emphasis>

CHROMagar has been reported to be useful for the rapid and accurate identification of Candida species. We tested 135 isolates of Candida species isolated from oropharyngeal candidiasis in HIV patients and found that it was useful in the presumptive identification of Candida albicans and Candida krusei. Occasional strains of C. tropicalis produced colonies with a greenish tinge making it difficult to differentiate from C. 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.     

Identification of <Emphasis Type="Italic">Candida</Emphasis> Species Isolated from Bovine Mastitic Milk and Their In Vitro Hemolytic Activity in Western Turkey

In this study, identification of 207 Candida isolates, previously isolated from mastitic bovine quarter milk samples at the level of genus, was made using API 20 C AUX system. The most frequently isolated species were Candida krusei (34.8%), followed by Candida rugosa (16.4%), Candida kefyr (12.6%), Candida albicans (10.1%), and Candida tropicalis (9.2%). Less common isolates were Candida zeylanoides (5.8%), Candida parapsilosis (4.3%), Candida guilliermondii (3.4%), Candida famata (1.9%), and Candida glabrata (1.5%). Additionally, in vitro hemolytic activity of all Candida strains were also examined in the present study. C. krusei (72 isolates), C. kefyr (26), C. albicans (21), C. tropicalis (19), C. zeylanoides (12), and C. glabrata (3) demonstrated both alpha and beta hemolysis at 48-h postinoculation. Only alpha hemolysis was detected in C. rugosa (34), C. guilliermondii (7), and C. famata (4), while C. parapsilosis (9) did not show any hemolytic activity after incubation for 72 h. Statistically significant difference (P < 0.001) was determined between the beta-hemolytic activities of Candida strains. The hemolytic activities of C. zeylanoides, C. albicans and C. kefyr were higher than other strains. This is the first study to describe variable hemolysis types exhibited by different Candida strains isolated from bovine mastitic milk in Turkey.     

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.     

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.     

Characterization,enzymatic activity and biofilm formation of Candida species isolated from goat milk

BackgroundData regarding yeast microbiota in goat milk is scarce.AimsTo isolate and identify species of the genus Candida in milk samples from clinically healthy goats, and evaluate their enzymatic activity and biofilm formation.Methods1092 milk samples from clinically healthy goats were collected and processed. The yeast isolates were identified by phenotypic, methods and their enzymatic activity (phospholipase, hemolysin and protease) and biofilm formation evaluated.ResultsWe obtained 221 Candida isolates belonging to six species: Candida kefyr (35.7%), Candida guilliermondii (33%), Candida famata (23.5%), Candida glabrata (5.9%), Candida albicans (1.35%) and Candida parapsilosis sensu lato (0.45%). Protease activity was detected in all Candida species while hemolysin activity was only present in C. kefyr, C. guilliermondii, C. famata and C. albicans. Only C. albicans showed phospholipase activity. With the exception of C. parapsilosis sensu lato, all Candida species formed biofilm, with 60.19% of the isolates being poor producers, 9.93% moderate producers, and 1.35% strong producers.ConclusionsThe milk of clinically healthy goats contains several species of the genus Candida that could play a role as opportunistic pathogens in mastitis.     

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.     

In Vitro Evaluation of Phospholipase,Proteinase, and Esterase Activities of <Emphasis Type="Italic">Candida parapsilosis</Emphasis> and <Emphasis Type="Italic">Candida metapsilosis</Emphasis>

The aim of this study is to characterize extracellular phospholipase, proteinase, and esterase activities of Candida parapsilosis and C. metapsilosis isolated from clinical sources. Using PCR-restriction fragment length polymorphism (PCR–RFLP) of the secondary alcohol dehydrogenase (SADH) gene fragment, we identified 20 as C. parapsilosis and 11 as C. metapsilosis from 31 isolates of C. parapsilosis species complex. No C. orthopsilosis was identified. A significantly high isolation frequency of C. metapsilosis (35.5%) was observed. Subsequent evaluation of enzymatic profile showed that 90.5% of C. parapsilosis and 91.7% of C. metapsilosis isolates were phospholipase producers. No difference in phospholipase activity was observed between two species. In terms of proteinase, 81.0% of C. parapsilosis and 83.3% of C. metapsilosis isolates were positive. A higher level of proteinase activity was detected in C. parapsilosis. A remarkably high proportion of both C. parapsilosis and C. metapsilosis isolates exhibited strong phospholipase and proteinase activities, suggesting that the production of these two enzymes might be common for them. On the other hand, both species similarly displayed rare esterase activity, with only one C. parapsilosis and two C. metapsilosis isolates being positive. Our data may further add to the confusion concerning the hydrolytic enzymatic activities of the C. parapsilosis complex, and a wider collection of isolates and standardized methods may help to address the issue.     

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.     

Evaluation of Antifungal Susceptibility Testing with Microdilution and Etest Methods of <Emphasis Type="Italic">Candida</Emphasis> Blood Isolates

Candida species that show an increasing number of clinical and/or microbiological resistance to several antifungals and are the most common agents of invasive fungal infections. The aim of this study was to investigate the in vitro susceptibility of Candida blood isolates to antifungal agents (amphotericin B, fluconazole, itraconazole, and voriconazole) by comparative use of the CLSI reference microdilution method and Etest. Four hundred Candida blood isolates (215 Candida albicans, 185 non-albicans Candida strains) were included in the study. The broth microdilution test was performed according to the CLSI M27 A2 document. Etest was carried out according to the manufacturer’s instructions. The MIC results obtained with reference microdilution were compared with those obtained with the Etest by using percent and categorical agreements. According to MIK₉₀ values, voriconazole was the most active and itraconazole was the least active drug in vitro against all Candida species. Other than voriconazole, statistically significant differences were found when the susceptibility of Candida albicans and non-albicans Candida spp. to amphotericin B, fluconazole, and itraconazole were compared. These antifungal agents were found to be more active to C. albicans. Among the non-albicans Candida species, the lowest MIC values were obtained for Candida parapsilosis isolates. When the standard method was compared with Etest, the total agreement was higher for C. albicans than for non-albicans species, especially for fluconazole and voriconazole. In view of the findings, it was concluded that itraconazole showed the lowest activity against all Candida species. Etest could be an alternative method in assessing the in vitro antifungal susceptibility of Candida spp., but it is more convenient to use the microdilution method for studying in vitro susceptibility of non-albicans species, in particular for those possessing high MIC values against azoles.     

DRBC agar: a new tool for <Emphasis Type="Italic">Candida dubliniensis</Emphasis> identification

Candida dubliniensis pathogenic species, which shares many phenotypic features with C. albicans, may be misidentified in the microbiology laboratory. The growth on DRBC agar at 25 °C was shown to be a new tool for differentiation between C. dubliniensis and C. albicans. All 27 isolates of C. dubliniensis showed in this medium rough colonies (peripheral hyphal fringes) and abundant chlamydospore production, while all 103 isolates of C. albicans showed smooth colonies without fringes or chlamydospores. DRBC agar allowed the differentiation of C. albicans from C. dubliniensis with 100 % sensitivity and specificity.     

Molecular Analysis of <Emphasis Type="Italic">Candida albicans</Emphasis> Isolates from Clinical Specimens

The aim of this study was to genotype Candida albicans strains isolated from various clinical specimens by using CA-INT-R and CA-INT-L primer pairs designed to span the region that includes the site of the transposable group-1 intron in the 25S rRNA gene. A total of 194 C. albicans isolates (28 invasive and 166 noninvasive) were genotyped. The frequencies of genotypes A, B, C and D were found as 51.0, 29.4, 19.1 and 0.5%, respectively. Statistically significant difference was determined between frequency of genotype distribution between invasive and noninvasive isolates (P < 0.001). Genotype C was more prevalent among invasive isolates while genotype A was in noninvasive ones. Furthermore, six different subtypes were determined among genotype A C. albicans isolates by restriction endonuclease analysis using a previously constructed differentiation scheme consisting of HaeIII and MspI digestions. This study demonstrated the genetic diversity of clinical isolates of C. albicans in our hospital.     

Heteroduplex mobility assay of the 26S rDNA D1/D2 region for differentiation of clinically relevant Candida species

The Heteroduplex Mobility Assay (HMA) method using the PCR amplified D1/D2 region of the 26S rDNA was tested for the differentiation of clinically relevant Candida species. Strains belonging to the same species are not expected to form heteroduplexes in this assay when their PCR products are mixed. D1/D2 HMA experiments between all Candida type strains tested showed heteroduplex formation, including Candida albicans and Candida dubliniensis. There was no heteroduplex formation when most clinical and non-type strains were tested against the type strain of their presumptive species, except when C. albicans WVE and C.␣dubliniensis TAI were analysed. Additional HMA experiments, phenotypic characterisation, and D1/D2 sequencing identified these isolates as Candida tropicalis and Candida parapsilosis, respectively. HMA provides a rapid and relatively simple molecular tool for the differentiation of potentially pathogenic Candida species.