Esterase activity of Candida species isolated from immunocompromised hosts

A total of 149 clinical isolates of Candida species isolated from immunocompromised patients were examined to ascertain their esterase activity by the Tween 80 opacity test, which is a biochemical test used mainly to differentiate between Candida albicans and Candida dubliniensis. Our results showed that C. albicans (92.3%), Candida tropicalis (92.3%), Candida parapsilosis (25%), C. dubliniensis (16.6%), Candida inconspicua (100%), and Candida lipolytica (100%) produced opacity halos through the 10-day post-inoculation period. The remaining Candida species did not produce a positive test response. These findings indicate that Tween 80 opacity test cannot be used as the sole phenotypic trait in the differentiation of C. albicans and C. dubliniensis.     

Phenotypic evaluation to differentiate Candida albicans from Candida dubliniensis

This study evaluated the phenotypic tests used to differentiate Candida albicans from Candida dubliniensis. A total of 55 isolates from vaginal secretions, oral cavity and hemoculture were studied. They were originally identified as C. albicans, based on their morphological and physiological characteristics. These isolates were tested for colony color development on CHROMagar Candida medium, growth at 45 degrees C on Sabouraud Dextrose agar, lipolytic activity on Tween 80 Agar medium and colony morphology and chlamydoconidia formation on Staib agar medium. Of the 55 isolates studied, seven yielded one or more phenotypic characteristics suggestive of Candida dubliniensis. These isolates were tested by PCR with specific primers for Candida dubliniensis and API ID 32. The seven isolates were confirmed as Candida albicans. All of these finding indicate that DNA based tests should be used for definitive identification of Candida dubliniensis.     

Lower filamentation rates of Candida dubliniensis contribute to its lower virulence in comparison with Candida albicans

Candida albicans and C. dubliniensis are very closely related yeast species. In this study, we have conducted a thorough comparison of the ability of the two species to produce hyphae and their virulence in two infection models. Under all induction conditions tested C. albicans consistently produced hyphae more efficiently than C. dubliniensis. In the oral reconstituted human epithelial model, C. dubliniensis isolates grew exclusively in the yeast form, while the C. albicans strains produced abundant hyphae that invaded and caused significant damage to the epithelial tissue. In the oral-intragastric infant mouse infection model, C. dubliniensis strains were more rapidly cleared from the gastrointestinal tract than C. albicans. Immunosuppression of Candida-infected mice caused dissemination to internal organs by both species, but C. albicans was found to be far more effective at dissemination than C. dubliniensis. These data suggest that a major reason for the comparatively low virulence of C. dubliniensis is its lower capacity to produce hyphae.     

Candida dubliniensis identification in Brazilian yeast stock collection

We investigated the presence of Candida dubliniensis among isolates previously identified as Candida albicans and maintained in a yeast stock collection from 1994 to 2000. All isolates were serotyped and further evaluated for antifungal susceptibility profile. After doing a screening test for C. dubliniensis isolates based on the capability of colonies to grow at 42 C, its final identification was obtained by randomly amplified polymorphic DNA (RAPD) analysis using three different primers. A total of 46 out of 548 screened isolates did not exhibit growth at 42 C and were further genotyped by RAPD. Eleven isolates were identified as C. dubliniensis with RAPD analysis. Regarding serotypes, 81.5% of C. albicans and all C. dubliniensis isolates belonged to serotype A. Of note, 9 out of 11 C. dubliniensis isolates were obtained from patients with acquired immunodeficiency syndrome (Aids) and all of them were susceptible to azoles and amphotericin B. We found 17 (3%) C. albicans isolates that were dose-dependent susceptibility or resistant to azoles. In conclusion, we found a low rate of C. dubliniensis isolates among stock cultures of yeasts previously identified as C. albicans. Most of these isolates were recovered from oral samples of Aids patients and exhibited high susceptibility to amphotericin B and azoles. C. albicans serotype A susceptible to all antifungal drugs is the major phenotype found in our stock culture.     

A molecular genetic system for the pathogenic yeast Candida dubliniensis

Candida dubliniensis is a recently described pathogenic yeast of the genus Candida that is closely related to Candida albicans but differs from it in several phenotypic and genotypic characteristics, including putative virulence traits, which may explain differences in the spectrum of diseases caused by the two species. In contrast to C. albicans, a molecular genetic system to study virulence of C. dubliniensis is lacking. We have developed a system for the genetic transformation of C. dubliniensis that is based on the use of the dominant selection marker MPA(R) from C. albicans that confers resistance to mycophenolic acid (MPA). Using this transformation system, a GFP (green fluorescent protein) reporter gene that was genetically engineered for functional expression in C. albicans and placed under control of the inducible C. albicans SAP2 (secreted aspartic proteinase) promoter was integrated into the C. dubliniensis genome. MPA-resistant transformants containing the SAP2P-GFP fusion fluoresced under SAP2-inducing conditions but not under SAP2-repressing conditions. These results demonstrate that the MPA(R) selection marker is useful for transformation of C. dubliniensis wild-type strains, that the GFP reporter gene is functionally expressed in C. dubliniensis, and that the C. albicans SAP2 promoter can be used for controlled gene expression in C. dubliniensis. These genetic tools will allow the dissection of the differences in virulence characteristics between the two pathogenic yeast species at the molecular level.     

Effect of farnesol on Candida dubliniensis morphogenesis

AIMS: Cell-cell signalling in Candida albicans is a known phenomenon and farnesol was identified as a quorum sensing molecule determining the yeast morphology. The aim of this work was to verify if farnesol had a similar effect on Candida dubliniensis, highlighting the effect of farnesol on Candida spp. morphogenesis. METHODS AND RESULTS: Two different strains of C. dubliniensis and one of C. albicans were grown both in RPMI 1640 and in serum in the presence of absence of farnesol. At 150 micromol l(-1) farnesol the growth rate of both Candida species was not affected. On the contrary, farnesol inhibited hyphae and pseudohyphae formation in C. dubliniensis. CONCLUSION: Farnesol seems to mediate cell morphology in both Candida species. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of farnesol on C. dubliniensis morphology was not reported previously.     

Cell surface hydrophobicity-associated adherence of Candida dubliniensis to human buccal epithelial cells

Microbial adherence to mucosal surfaces is an important first step in the initiation of the pathogenic process in the oral cavity. Candida albicans, the most adherent and pathogenic Candida species, utilizes a variety of mechanisms to adhere to human tissues. Although the strongest mechanism of adherence involves mannoprotein adhesins on C. albicans, cell surface hydrophobicity (CSH) plays an important role in the adherence process by providing hydrophobic interactions that turn the initial attachment between the yeast and a surface into a strong bond. Recent cell wall analytical and comparative studies showed that, Candida dubliniensis, unlike C. albicans, possesses cell surface variations that allow it to be constantly hydrophobic, regardless of growth temperature. Based on these observations, the present study was designed to compare the adherence abilities of C. dubliniensis and C. albicans to pooled human buccal epithelial cells (BEC), in regards to their cell surface hydrophobicity. Ten C. albicans and nine C. dubliniensis isolates, as well as the C. albicans hydrophobic variant A9V10 were evaluated for adherence with BEC using visual aggregation in the wells of a microtiter plate and microscopic examination. All 11 C. albicans isolates failed to show adherence to BEC, visually or microscopically, when grown at 37 degrees C. The same isolates, however, showed significant increase in aggregation and microscopic adherence to BEC when grown at 25 degrees C. All C. dubliniensis isolates tested and the A9V10 C. albicans hydrophobic variant resulted in visual aggregation and adhered to BEC when grown at either temperature. The findings from this study show that, based on comparative adherence results and growth temperature changes, C. dubliniensis seems to have greater adherence to BEC than do typical C. albicans strains and that hydrophobic interactions seem to be the mechanism of adherence involved. Although many questions remain to be answered regarding the clinical implications of this observed in vitro enhanced adherence of C. dubliniensis to human BEC, these findings support the establishment of this novel species as a clinically significant yeast.     

Discrimination between<Emphasis Type="Italic">Candida albicans</Emphasis> and<Emphasis Type="Italic">Candida dubliniensis</Emphasis> isolated from HIV-positive patients by using commercial method in comparison with PCR assay

Nineteen clinical isolates of Candida albicans and C. dubliniensis were isolated from patients (majority of them HIV-positive) in Slovakia, Brazil, Thailand and Japan. Species discrimination was performed by using growth on CHROMagar Candida, commercial biochemical set API 20C AUX, germ-tube test in human serum, growth at 42 and 45 degrees C on Sabouraud-dextrose agar as well as on CHROMagar Candida, assimilation of D-xylose and methyl alpha-D-glucoside by glass-tube test, and production of chlamydospores. These tests were completed by PCR using Cd-oligo2/F and Cd-oligo2/R primer pair specific for C. dubliniensis. Six clinical isolates were confirmed to be C. dubliniensis, remaining 13 strains were determined as C. albicans. The use of conventional method showed that the determination is markedly influenced by personal evaluation suggesting the necessity of using the combination of many tests to obtain correct results comparing with accurate and rapid PCR assay. For discrimination between C. albicans and C. dubliniensis we recommend the combination of primo-cultivation on CHROMagar, followed by germ-tube test and PCR.     

Calcineurin controls drug tolerance, hyphal growth, and virulence in Candida dubliniensis

Candida dubliniensis is an emerging pathogenic yeast species closely related to Candida albicans and frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during C. dubliniensis infection is common and constitutes a significant therapeutic challenge. The calcineurin inhibitor FK506 exhibits synergistic fungicidal activity with azoles or echinocandins in the fungal pathogens C. albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In this study, we show that calcineurin is required for cell wall integrity and wild-type tolerance of C. dubliniensis to azoles and echinocandins; hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast to C. albicans, in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that calcineurin and Crz1 play a clearly demonstrable role in hyphal growth in response to nutrient limitation in C. dubliniensis. We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin, in C. dubliniensis. Similar to C. albicans, C. dubliniensis calcineurin enhances survival in serum. C. dubliniensis calcineurin and crz1/crz1 mutants exhibit attenuated virulence in a murine systemic infection model, likely attributable to defects in cell wall integrity, hyphal growth, and serum survival. Furthermore, we show that C. dubliniensis calcineurin mutants are unable to establish murine ocular infection or form biofilms in a rat denture model. That calcineurin is required for drug tolerance and virulence makes fungus-specific calcineurin inhibitors attractive candidates for combination therapy with azoles or echinocandins against emerging C. dubliniensis infections.     

Identification of Candida albicans using the chromogenic medium Albicans ID

The correct identification of the microrganism is the base for epidemiological studies and treatment of infections. The aim of our study was to evaluate the efficacy of the chromogenic media Albicans ID (bioMerieux, France) in the identification of Candida albicans. A total of 190 yeasts strains were evaluated in the study. A rate of 100% of all C. albicans (80) and Candida dubliniensis (five) strains exhibited blue color. Nevertheless, the blue color was also observed with cultures of Candida rugosa (3/5) and Candida tropicalis (3/17). Albicans ID cromogenic media presented specificity rate of 90% and positive and negative predictive values of 88% and 100%, respectively, in the identification of C. albicans.     

Comparison of the epidemiology, drug resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans

Candida dubliniensis is a pathogenic yeast species that was first identified as a distinct taxon in 1995. Epidemiological studies have shown that C. dubliniensis is prevalent throughout the world and that it is primarily associated with oral carriage and oropharyngeal infections in human immunodeficiency virus (HIV)-infected and acquired immune deficiency syndrome (AIDS) patients. However, unlike Candida albicans, C. dubliniensis is rarely found in the oral microflora of normal healthy individuals and is responsible for as few as 2% of cases of candidemia (compared to approximately 65% for C. albicans). The vast majority of C. dubliniensis isolates identified to date are susceptible to all of the commonly used antifungal agents, however, reduced susceptibility to azole drugs has been observed in clinical isolates and can be readily induced in vitro. The primary mechanism of fluconazole resistance in C. dubliniensis has been shown to be overexpression of the major facilitator efflux pump Mdr1p. It has also been observed that a large number of C. dubliniensis strains express a non-functional truncated form of Cdr1p, and it has been demonstrated that this protein does not play a significant role in fluconazole resistance in the majority of strains examined to date. Data from a limited number of infection models reflect findings from epidemiological studies and suggest that C. dubliniensis is less pathogenic than C. albicans. The reasons for the reduced virulence of C. dubliniensis are not clear as it has been shown that the two species express a similar range of virulence factors. However, although C. dubliniensis produces hyphae, it appears that the conditions and dynamics of induction may differ from those in C. albicans. In addition, C. dubliniensis is less tolerant of environmental stresses such as elevated temperature and NaCl and H(2)O(2) concentration, suggesting that C. albicans may have a competitive advantage when colonising and causing infection in the human body. It is our hypothesis that a genomic comparison between these two closely-related species will help to identify virulence factors responsible for the far greater virulence of C. albicans and possibly identify factors that are specifically implicated in either superficial or systemic candidal infections.     

Candida dubliniensis and Candida albicans display surface variations consistent with observed intergeneric coaggregation

Adherence of yeasts to other microorganisms and epithelial cell surfaces is important in their colonization. Comparative studies based on the coaggregation of Candida dubliniensis versus Candida albicans with Fusobacterium nucleatum and other oral bacteria suggested differences in the surfaces of these yeasts. Transmission electron microscopy was used to test the hypothesis that there are morphologic variations in the cell surface of these two species. C. dubliniensis type strain CD36 and C. albicans ATCC 18804 were grown on Sabouraud's dextrose agar at various growth temperatures. In some experiments suspensions of yeast cells were treated with dithiothreitol. Fixation for transmission electron microscopy was accomplished using dimethylsulfoxide and alcian blue added to 3% paraformaldehyde and 1% glutaraldahyde in cacodylate buffer. The cell wall of both species was predominantly electron lucent and was visibly differentiated into several layers. A thin electron dense outer layer was seen with clearly visible fibrillar structures, closely associated to the cytoplasmic membrane. The length of the fibrils of the C. albicans cells grown at 37 degrees C was approximately two times greater than those of the cells grown at 25 degrees C. The fibrils of the 37 degrees C-grown cells were thin, distinct and tightly packed whereas those of the 25 degrees C-grown cells appeared blunt, loosely spaced and aggregated. C. dubliniensis demonstrated short, blunt fibrils appearing similar to those of the 25 degrees C-grown C. albicans cells. C. dubliniensis showed no difference in the density, length and arrangement of fibrils between the 25 degrees C and 37 degrees C growth temperatures. The shortest and most aggregated fibrils seen were of the 45 degrees C-grown C. albicans cells. Dithiothreitoltreated 37 degrees C-grown C. albicans cells revealed a distorted and partially destroyed fibrillar layer. In this investigation C. dubliniensis, unlike C. albicans, displayed an outer fibrillar layer that did not vary with variations in growth temperature. In addition, the fibrils on the C. dubliniensis cells were similar to those of the 25 degrees C-grown C. albicans in that they were considerably shorter and less dense than those of the 37 degrees C-grown C. albicans cells. It can be postulated, that C. dubliniensis exhibits constant cell surface characteristics consistent with hydrophobicity and that this property may give this species an ecological advantage. Therefore, C. dubliniensis may compete well in oral environments via enhanced attachment to oral microbes and other surfaces, perhaps even more efficiently than C. albicans.     

26S rDNA单链构象多态性分析在临床酵母菌菌种鉴定中的应用

摘要： 【目的】探讨酵母菌临床分离株26S rDNA D1/D2区序列种内相似性和种间差异性的快速检测方法,为临床酵母菌菌种鉴定方法的改进奠定基础。调查北京地区临床酵母菌的种群多样性,为国内酵母菌感染的流行病学研究提供新的基础数据。【方法】用5种常见临床酵母菌种的模式和权威菌株作为标准参考菌株,从北京四家综合性医院收集临床酵母菌260余株,PCR扩增其26S rDNA D1/D2区,对扩增产物进行单链构象多态性（Single-Strand Conformation Polymorphism,SSCP）分析和序列测定分析。【结果】常见病原酵母菌26S rDNA D1/D2区的SSCP图谱具有明显的种间差异性和种内相似性,可以通过该方法对菌株进行初步的菌种鉴定。D1/D2-SSCP和序列分析相结合,对260余株临床酵母菌进行了菌种鉴定,共鉴定有10个属20个种,优势属为念珠菌属(Candida),优势种及其所占比例分别是：C. albicans (57.7%), C. parapsilosis (10.0%), C. tropicalis (9.2%), C. glabrata (6.7%)和C. krusei (5.8%),并发现过去从未或很少报道致病的酵母菌种,愈来愈多地出现在临床分离菌株中。【结论】 26S rDNA D1/D2区的SSCP图谱分析为临床酵母菌株的快速鉴定提供了新的方法;北京地区酵母菌临床分离株呈种群多样性分布,C. albicans虽然仍占优势,但其它念珠菌种的比例已达42%。     

Specific antibody response in a patient with Candida dubliniensis fungemia

We report a case of fungemia caused by Candida dubliniensis in a non-HIV infected patient. Multiple cultures of blood performed over a period of 13 days were positive for this recently described yeast species. The C. dubliniensis isolates recovered were susceptible to fluconazole in vitro and the patient responded to intravenous therapy with this antifungal agent. It was possible to differentiate the fungemia caused by C. dubliniensis in this patient from that caused by C. albicans in other patients on the basis of the analysis of the antibody response since the C. dubliniensis-infected patient exhibited a characteristic and specific antibody response against a cell wall component of 160-170 kDa.     

Isogenic strain construction and gene targeting in Candida dubliniensis

Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans but differs from it with respect to epidemiology, certain virulence characteristics, and the ability to develop fluconazole resistance in vitro. A comparison of C. albicans and C. dubliniensis at the molecular level should therefore provide clues about the mechanisms used by these two species to adapt to their human host. In contrast to C. albicans, no auxotrophic C. dubliniensis strains are available for genetic manipulations. Therefore, we constructed homozygous ura3 mutants from a C. dubliniensis wild-type isolate by targeted gene deletion. The two URA3 alleles were sequentially inactivated using the MPA(R)-flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. The URA3 gene from C. albicans (CaURA3) was then used as a selection marker for targeted integration of a fusion between the C. dubliniensis MDR1 (CdMDR1) promoter and a C. albicans-adapted GFP reporter gene. Uridine-prototrophic transformants were obtained with high frequency, and all transformants of two independent ura3-negative parent strains had correctly integrated the reporter gene fusion into the CdMDR1 locus, demonstrating that the CaURA3 gene can be used for efficient and specific targeting of recombinant DNA into the C. dubliniensis genome. Transformants carrying the reporter gene fusion did not exhibit detectable fluorescence during growth in yeast extract-peptone-dextrose medium in vitro, suggesting that CdMDR1 is not significantly expressed under these conditions. Fluconazole had no effect on MDR1 expression, but the addition of the drug benomyl strongly activated the reporter gene fusion in a dose-dependent fashion, demonstrating that the CdMDR1 gene, which encodes an efflux pump mediating resistance to toxic compounds, is induced by the presence of certain drugs.     

Comparison of Candida dubliniensis and C. albicans based on polar lipid composition

AIMS: To test the hypothesis that strains of Candida dubliniensis and C. albicans can be differentiated on the basis of polar lipid profiles. METHODS AND RESULTS: Five isolates of C. dubliniensis and six isolates of C. albicans were tested by growth at 45 degrees C, production of chlamydospores on cornmeal agar, colonial colour on CHROMagar Candida medium and assimilation of DL-lactate, alpha-methyl-D-glucoside and xylose. Polar lipids were then extracted from freeze-dried cultures and analysed using fast atom bombardment mass spectrometry. Isolates were grouped by single linkage clustering based on correlation coefficients for strain pairs calculated with carboxylate and phospholipid molecular species distributions. The most intense carboxylate and phospholipid molecular species anions were of m/z 281 (C(18 : 1)) and m/z 515 (PA 23 : 2). Phosphatidylethanolamine and phosphatidylglycerol were the predominant phospholipid families in C. dubliniensis, compared with phosphatidic acid in C. albicans isolates. All of the C. dubliniensis isolates grouped together in one cluster, whereas all of the C. albicans isolates grouped in a separate cluster. CONCLUSIONS: Fast atom bombardment mass spectrometry can differentiate the two species based on analysis of polar lipid distributions. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings demonstrate that C. dubliniensis and C. albicans have distinct polar lipid profiles.     

The presence of fluconazole-resistant Candida dubliniensis strains among Candida albicans isolates from immunocompromised or otherwise debilitated HIV-negative Turkish patients

The newly described species Candida dubliniensis phenotipically resembles Candida albicans in many respects and so it could be easily misidentified. The present study aimed at determining the frequency at which this new Candida species was not recognized in the authors' university hospital clinical laboratory and to assess antifungal susceptibility. In this study, six identification methods based on significant phenotypic characteristics each proposed as reliable tests applicable in mycology laboratories for the differentiation of the two species were performed together to assess the clinical strains that were initially identified as C. albicans. Only the isolates which have had the parallel results in all methods were assessed as C. dubliniensis. One hundred and twenty-nine C. albicans strains isolated from deep mycosis suspected patients were further examined. Three of 129 C. albicans (2 from oral cavity, 1 from sputum) were reidentified as C. dubliniensis. One of the strains isolated from oral cavity and that from the sputum were obtained at two months intervals from the same patient with acute myeloid leukemia, while the other oral cavity strain was obtained from a patient who had previously been irradiated for a laryngeal malignancy. Isolates were all susceptible in vitro to amphotericin B, with the MIC range 0.125 to 0.5 &mgr;g/ml, resistant to fluconazole, with MICs >/=64 &mgr;g/ml, and resistant to ketoconazole, with MICs >/=16 &mgr;g/ml, dose-dependent to itraconazole with a MIC range 0.25-0.5 &mgr;g/ml, and susceptible to flucytosine, with a MIC range 1-4 &mgr;g/ml.     

Differences in extracellular enzymatic activity between Candida dubliniensis and Candida albicans isolates

Twenty-six Candida dubliniensis and 27 Candida albicans oral strains isolated from patients infected by the human immunodeficiency virus (HIV) were tested for germ tube production and 21 extracellular enzymatic activities. Assessment of the enzymatic profile was performed by using the API-ZYM commercial kit system (bioMerieux, France), which tests 19 different enzymes. Protease activity was expressed during the first days of incubation by 100% of the strains studied and resulted higher than phospholipase activity in the C. dubliniensis and C. albicans strains tested. The API-ZYM profile of the C. dubliniensis and C. albicans strains differs with respect to the number and percentage of the enzymes considered, as well as with the intensity of the substrate metabolized by the strains, in particular for the enzymes n 8 (cystine-arylamidase), n 12 (naphtol-AS-BI-phosphohydrolase) and n 16 (alpha-glucosidase). These enzymes may be useful to differentiate C. dubliniensis and C. albicans together with other phenotypic characteristics proposed in the literature. No relationship among protease, phospholipase and other extracellular enzymatic activities was observed in C. dubliniensis. The average percentage of strains filamentation after 4 h was between 32 and 42%.