Doxorubicin selects for fluconazole-resistant petite mutants in Candida glabrata isolates

Candida infections are a permanent threat to immunocompromised individuals such as cancer patients, and Candida glabrata has emerged as a major problem in recent years. Resistance may develop during lengthy antifungal therapies and is often mediated by upregulation of fungal drug efflux pumps. During chemotherapy the yeast cell is also exposed to cytotoxic agents that may affect its drug susceptibility. Four C. glabrata isolates, three susceptible and one resistant to fluconazole (FLU), were incubated with 20 μg/ml of doxorubicin (DOX) for 90 min. In a second experiment, the isolates were cultured with DOX for ten days. Samples were taken on subsequent days to determine the minimal inhibitory concentration (MIC) of FLU and to analyze expression of CgCDR1, CgCDR2, CgSNQ2 and CgPDR1. Samples were also used to assess the petite phenotype. Short-term DOX exposure did not induce efflux pump gene expression, but genes were consistently overexpressed in FLU-susceptible isolates during long-term exposure. An increase in MIC values on day 6 in two of the isolates coincided with the first occurrence of petite mutants in all susceptible isolates. The respiratory deficiency of selected petite mutants was confirmed by culturing mutants on agar containing glycerol as the sole carbon source. FLU MIC values for respiratory-deficient clones were ≥64 μg/ml, and efflux pump gene expression was greatly increased. The resistant isolate did not develop mitochondrial dysfunction. In summary, the cytotoxic agent DOX selects for FLU-resistant respiratory-deficient C. glabrata mutants, which may affect antifungal therapy.     

Nosocomial Candida glabrata Colonization: an Epidemiologic Study

Candida glabrata has emerged as an important nosocomial pathogen, yet little is known about its epidemiology. We prospectively followed 98 patients admitted to a medical intensive care unit and the bone marrow transplant unit of a university hospital. Samples from environmental surfaces and the hands of hospital personnel were also cultured. Patients with newly acquired C. glabrata strains were compared to controls who were culture negative for C. glabrata. C. glabrata was recovered from multiple sites from 24 patients and three environmental surfaces. Sixteen patients (17%) acquired C. glabrata after admission to the study units. Significant risk factors for the nosocomial acquisition of C. glabrata were prolonged duration of hospitalization in the unit and prior antimicrobial use. Strain delineation by restriction enzyme analysis revealed 28 different strains of C. glabrata; three strain types were common to nine patients. The environmental isolates were of the same strain type and common to five patients (four patients with newly acquired strains). These results suggest the possibility of exogenous nosocomial acquisition of C. glabrata, including the possible acquisition from the hospital environment. Transmission may be by indirect contact since identical strains of C. glabrata were recovered from patients who were geographically and temporally associated.     

Detection of fluconazole-resistant isolates of Candida glabrata by using an agar screen assay

The ability of a fluconazole-containing agar screen assay to accurately detect isolates of Candida glabrata resistant to the azole antifungal agent fluconazole was evaluated on a collection of 100 clinical isolates of this organism. Results were correlated with the MIC of fluconazole for these isolates and compared with the results of a previously published disk diffusion-based fluconazole resistance screening test. Agar screen assay results were in categorical agreement with MIC-based determinations for 97% (97/100) of the isolates tested. This correlation was higher than that obtained with the disk diffusion technique, which categorized only 87% (87/100) of isolates correctly, and suggests that the agar screening approach can effectively expedite fluconazole susceptibility testing of C. glabrata isolates.     

Techniques for investigation of an apparent outbreak of infections with Candida glabrata.

A cluster of Candida glabrata isolates recovered from seven patients in an intensive care unit over a 10-week period were compared with a collection of isolates from six epidemiologically distinct outpatients and a reference strain by several DNA typing methods. Restriction enzyme analysis with HinII distinguished 13 strains from the 14 sources and was the method of choice. Pulsed-field gel electrophoresis and random amplification of polymorphic DNA both detected nine types from the 14 sources; however, the results of these two methods did not always correlate. These methods demonstrated that five of the seven patients had distinguishable strains and that cross-infection was unlikely.     

Fournier's gangrene due to Candida glabrata.

Fournier's gangrene is a rare and serious event. Usual pathogens are bacteria of the skin and the digestive tract. Candida species are exceptionally involved, mostly Candida albicans. We report a patient with non-C. albicans Candida sp. Fournier's gangrene who survived with an appropriate antifungal therapy. Yeasts should be considered as emerging pathogens in pelvic infections due to the increase in long-term immunocompromised patients.     

Production of indole pigments by Candida glabrata.

When provided as the sole nitrogen source tryptophan induces the production of several indole alkaloids, e.g., pityriacitrin, malasseziaindole, pityriaanhydride and pityriarubin with proven biological activity in the lipophilic yeast Malassezia furfur. So far these pigments seem to have been unique and only produced by highly specialized basidiomycetal yeasts of the genus Malassezia. Having surprisingly observed a brown pigmented Candida glabrata isolate as a contaminant on such a pigment inducing culture plate, we systematically analyzed whether this ascomycetal yeast can also synthesize the respective pigments. Therefore, 30 Candida glabrata strains, including the ex-type strain CBS 138, were cultured for 2 weeks on a pigment-inducing medium containing L-tryptophan. This culture medium along with the resultant biomass was then extracted with ethyl acetate. The extracted pigments were separated into six fractions by column chromatography. Each of these fractions was subjected to thin-layer chromatography (TLC) on silica gel and yielded identical pigment bands comparable to those observed with M. furfur. In the case of strain CBS 138, the individual TLC zones were further purified by HPLC and structural analysis of the pure metabolites was performed by mass spectrometry and proton nuclear magnetic resonance ((1)H-NMR), thereby proving the presence of pityriacitrin, malassezia indole, pityriaanhydride and pityriarubin C. Since lineage divergence of Basidiomycota and Ascomycota occurred approximately 600 million years ago, our findings demonstrate that the complex underlying biochemical pathway has not been exclusively evolved in the highly adapted basidiomycetes yeast M. furfur, but instead seems to be rather fundamental and archaic. Therefore, further investigations on the potential biological properties and the genetic regulation of these metabolites are needed to elucidate their hitherto unknown functions.     

Restriction fragment length polymorphism analysis of azole-resistant and azole-susceptible Candida albicans strains.

Restriction fragment length polymorphism analysis was performed with the endonucleases EcoRI, BglII, and HinfI on a collection of Candida albicans strains comprising eight strains randomly selected from clinical microbiology laboratory specimens, three reported azole-resistant strains from treatment failures, and several subcultures of the azole-resistant strain NCPF 3310 (also known as the Darlington strain) received from different laboratories. The results demonstrated a diversity of the restriction fragment length polymorphism patterns that were obtained and revealed that two of the proposed Darlington subcultures had patterns distinct from each other and from those of the other Darlington isolates; both were also found to have lost their azole resistance.     

双重实时荧光定量PCR法鉴定克柔念珠菌和光滑念珠菌

目的 建立一种快速、灵敏、特异的鉴定克柔念珠菌和光滑念珠菌的双重实时荧光定量PCR方法.方法 以核糖体基因内转录间隔区Ⅱ(ITSⅡ)为靶目标,设计并合成分别针对克柔念珠菌、光滑念珠菌的种特异引物和探针.建立双重实时荧光定量PCR反应体系,并用该体系对呼吸道相关致病菌进行检测.鉴定结果与临床常规鉴定方法对照,评价其敏感度、特异度及重复性.结果 通过对100例样品的检测,结果显示该双重实时荧光定量PCR法检测标本的鉴定结果与常规鉴定方法的结果对照,特异度为100%,敏感度为100%;最小能检测到10个拷贝数的重组质粒;批内重复实验和批间重复实验结果均与常规鉴定方法结果相符.结论 双重荧光定量PCR法鉴定克柔念珠菌和光滑念珠菌,特异度和敏感度高,重复性好,且快速、简便,该方法将有助于念珠菌病的早期诊断和针对性治疗.     

Detection of human P-glycoprotein-like molecule in azole-resistant Candida albicans from HIV+ patients

Azole resistance in Candida albicans may be due to several mechanisms. It has been demonstrated that C. albicans possesses sequences with a high degree of homology with the human MDR-1 gene coding for P-glycoprotein (P-gp), belonging to the ATP-binding cassette transporter (ABC) superfamily and responsible for the multidrug resistance (MDR) in tumor cells. On this basis, the expression and intracellular localization of human P-gp-like molecule in C. albicans strains showing different sensitivity to fluconazole were investigated by flow cytometry and immunoelectron microscopy. Post-embedding immunolabeling revealed that monoclonal antibody (mAb) MM4.17, which recognizes an external epitope of human P-gp, reacted with both fluconazole-sensitive (3153 and CO 23-1) and fluconazole-resistant (AIDS 68 and CO 23-2, isolated from AIDS patient and in vitro drug-selected, respectively) strains of C. albicans. However, the resistant strains displayed a number of MM4.17-reactive epitopes much higher than the drug-sensitive ones. The C. krusei ATCC 6458 strain, whose resistance is not mediated by the presence of ABC transporters, was not reactive at all with mAb MM4.17. The specificity of the immunolabeling was confirmed by a competitive inhibition assay performed by using phage clone particles capable of mimicking the MM4.17-reactive epitope. The flow cytometric analysis confirmed a higher level of intracytoplasmic P-gp expression in azole-resistant strains of C. albicans. Both cyclosporin A and verapamil, which are well-known MDR inhibitors, strongly reduced the MICs for fluconazole and itraconazole of the tested azole-resistant AIDS 68 strain, while they did not influence the MICs of either the sensitive 3153 strain of C. albicans or the ATCC 6458 strain of C. krusei. Overall, our data suggest the existence of a P-gp-like drug efflux pump in C. albicans that may participate in the mechanisms of azole-resistance of this fungus.     

Candida glabrata fungaemia in intensive care units.

Candidaemia is increasingly important in intensive care units (ICUs). Compared with Candida albicans fungaemia, the impact of C. glabrata fungaemia on ICU patients is not well-known. The aim of this study was to investigate the clinical features, the antifungal susceptibility and the treatment outcomes of C. glabrata fungaemia in ICU patients. The medical records of ICU patients with candidaemia between 2000 and 2005 were reviewed retrospectively, and antifungal susceptibility testing was performed for isolates of C. glabrata. Among 147 episodes of candidaemia occurring in adult ICUs, C. glabrata was the second most common species and accounted for 45 (30%) episodes of candidaemia. The incidence of C. glabrata fungaemia was 1.3/1000 ICU admissions. Fluconazole resistance was found in 11% of C. glabrata isolates. The 30-day all-cause mortality rate was 58%. Therapeutic regimens containing amphotericin B were associated with better outcome. Despite higher fluconazole resistance, C. glabrata candidaemia was not associated with greater mortality than non-glabrata candidaemia in the ICU setting.     

Candida glabrata strain relatedness by new microsatellite markers

We investigated six microsatellite markers to type 85 unrelated and 118 related isolates of Candida glabrata from 36 patients. Three new markers were selected from the complete sequence of CBS138 and three previously described markers, RPM2, MTI and ERG3 were used. We found a genetic diversity of 0.949 by combining four of them. By applying the new microsatellite markers GLM4, GLM5 and GLM6 we were able to discriminate 29 isolates, originally identified by the more established markers, RPM2, MTI and ERG3. When epidemiologically closely related isolates from 36 patients were typed, 25 patients (72%) exhibited identical or highly related multilocus genotypes. We noted a microvariation in 4 of the patients. This minor change of one locus could be explained by a single step mutation. Since one of these patients had not received antifungal treatment; thus, the relationship between genome variation and antifungal therapy remains controversial. We can conclude from our analysis of these new microsatellite markers that they are highly selective and therefore should be considered as a useful typing system for differentiating related and unrelated isolates of C. glabrata, as well as being able to detect microvariation.     

Isolation of mutants of Candida glabrata resistant to miconazole

Elucidation of the mode of action of azole antifungals would be aided by studying resistant mutants. It is difficult to obtain mutants of Candida albicans in the laboratory, and there have only been a few studies on clinical isolates which seem to be resistant because of impaired drug uptake. C. glabrata, unlike C. albicans, is haploid and more likely to give rise to resistant variants. Over 30 mutants have been isolated by selection with miconazole on solid medium and have MICs of miconazole about ten times that of the parental strain. One such mutant has a reduced growth rate and final cell yield. In intact cells, ergosterol biosynthesis is tenfold less sensitive to miconazole than in the parent. However, uptake of [3H]miconazole by cells is identical in both strains. The significance of these observations is discussed.     

Non-Utilization of sucrose by the petite mutant of a distiller's yeast

Summary A number of yeast strains are known to be unable to metabolize several sugars (galactose, maltose, -methylglucoside) when converted to their petite mutants. The basis of this phenomenon is considered to be the loss of the ability to transport the sugars across the cell membrane. However, sucrose is believed to be hydrolyzed before the products are transported into the cell, and the enzyme responsible (invertase) is thought to be either present in the periplasmic space or to be bound to the outer surface of the cytoplasmic membrane. Hence the loss of the ability to metabolize sucrose may infer the impairment of the mechanism for transport of invertase to its normal location outside the cytoplasm. We have found a distiller's yeast strain which has lost the ability to metabolize sucrose when it is converted to the petite mutant, and we report here some of its properties. We have shown that the cell produces invertase, which is present in the cell-free extract, but not in the pellet of cell walls and unbroken cells, though we have not determined whether the enzyme is present in the cytoplasm in the glycosylated or the unglycosylated form. The ability of the strain to ferment sucrose is also impaired in respiratory-competent cells, when the determination is made under anaerobic conditions.     

Investigation of the Function of Candida albicans Als3 by Heterologous Expression in Candida glabrata

During hematogenously disseminated infection, blood-borne Candida albicans invades the endothelial cell lining of the vasculature to invade the deep tissues. Although the C. albicans Als3 invasin is critical for invasion and damage of endothelial cells in vitro, a C. albicans als3Δ/Δ mutant has normal virulence in the mouse model of disseminated infection. We hypothesized that the contribution of Als3 to virulence is obscured by the presence of additional C. albicans invasins. To elucidate the in vivo function of Als3, we heterologously expressed C. albicans ALS3 in Candida glabrata, a yeast that lacks a close ALS3 ortholog and has low virulence in mice. We found that following intravenous inoculation into mice, the ALS3-expressing strain preferentially trafficked to the brain, where it induced significantly elevated levels of myeloperoxidase, tumor necrosis factor, monocyte chemoattractant protein 1, and gamma interferon. Also, the ALS3-expressing strain had enhanced adherence to and invasion of human brain microvascular endothelial cells in vitro, demonstrating a potential mechanism for ALS3-mediated neurotropism. In addition, upon initiation of infection, the ALS3-expressing strain had increased trafficking to the cortex of the kidneys. With prolonged infection, this strain persisted in the kidneys at significantly higher levels than the control strain but did not induce an elevated inflammatory response. Finally, the ALS3-expressing strain had increased resistance to neutrophil killing in vitro. These results indicate that during disseminated infection, Als3 mediates initial trafficking to the brain and renal cortex and contributes to fungal persistence in the kidneys.     

Influence of growth conditions on cell surface hydrophobicity of Candida albicans and Candida glabrata.

The effect of cultural conditions on cell surface hydrophobicity of Candida albicans and Candida glabrata was tested. C. albicans cells grown at room temperature were more hydrophobic than cells grown at 37 degrees C. No consistent pattern was observed with C. glabrata. Relative hydrophobicity was found to vary with the growth phase and growth medium for both species. The implications for pathogenesis studies are discussed.     

Rapid discrimination between Candida glabrata, Candida nivariensis, and Candida bracarensis by use of a singleplex PCR

We report here a PCR-based assay using a single primer pair targeting the RPL31 gene that allows discrimination between Candida glabrata, Candida bracarensis, and Candida nivariensis according to the size of the generated amplicon.     

Screening protocol for Torulopsis (Candida) glabrata.

A screening test has been developed for the presumptive identification of Torulopsis (Candida) glabrata from other common clinical isolates of yeast-like fungi. An interlaboratory comparison of a protocol consisting of morphology on cornmeal Tween 80 agar and trehalose fermentation at 42 degrees C was successful in differentiating T. glabrata from other taxa that are frequent or possible clinical isolates. The screening results for 517 clinical yeast isolates, 241 of which were T. glabrata, were compared with their final identification via commercial systems (API20C Yeast Identification System [bioMERIEUX, Hazelwood, Mo.] and Rapid Yeast Identification Panel [Dade Microscan, Sacramento, Calif.]). The trehalose screening test has a sensitivity and a specificity of 97.8 and 95.8%, respectively, and a positive predictive value of 97.4% and a negative predictive value of 96.5%. Overall, the trehalose screen had an efficiency rating of 93.9% for ruling in or out T. glabrata. Since T. glabrata represents a substantial part of the workload in a clinical laboratory, a significant reduction in direct and indirect costs should be realized.