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Infections with Candida glabrata have recently gained worldwide attention owing to its association with long hospitalizations and high mortality rates. This problem is highlighted when the infection is associated with echinocandin resistance, which is used for first-line therapy. Echinocandin resistance is exclusively attributed to functional mutations in FKS genes, and especially in hot spot (HS) regions. Unfortunately, few studies have focused on the rapid identification of FKS mutations associated with echinocandin resistance in C. glabrata. This study was intended to evaluate and validate the use of Surveyor nuclease assay (SN) for detection of FKS gene mutations. SN was evaluated against three segments of FKS1 and FKS2 genes including whole gene, regions including all HSs, and the region including only HS1. Our results showed that SN results are basically dependent on the type of gene as well as the segment type. Interestingly, SN can detect mutations in the region containing HS1 in both FKS1 and FKS2 genes. Furthermore, SN can detect mutations in the segment containing all HS regions for FKS1 but not FKS2. SN was unable to detect mutations in the whole FKS1 and FKS2 genes. As far as we know, this is the first study to validate SN for rapid identification of FKS gene mutations. This assay could be used as a sample for rapid identification of mutations associated with HS1 region in FKS genes, which have a predominant role for echinocandin resistance induction in C. glabrata. [ABSTRACT FROM AUTHOR]

Highlights • The recombinant enzyme CgHMGR was used as a model to test new compounds with possible antifungal activity. • Compounds 3 and 4 inhibited CgHMGR enzyme activity, with an IC 50 lower than that for α-asarone. • Molecular docking studies revealed that compounds 3 and 4 bind to CgHMGR active site. • Effect caused by simvastatin and α-asarone-based compounds were reversed with the addition of sterols. Abstract Due to increasing resistance of Candida species to antifungal drugs, especially azoles, new drugs are needed. The proposed compounds 3 and 4 are analogous to α-asarone (2) , a naturally occurring potent inhibitor of HMGR with hypolipidemic and antifungal activity. We used the recombinant enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase of Candida glabrata (CgHMGR) as a model to test the effectiveness of the test compounds. Compounds 3 and 4 demonstrated inhibitory kinetics, having lower IC 50 values (42.65 μM and 28.77 μM, respectively) than compound 2 (>100 μM). The docking studies showed better binding energies for compounds 3 and 4 (−5.35 and −6.1 kcal/mol, respectively) than for compound 2 (−4.53 kcal/mol). These findings suggest that the tested compounds are better than their natural analogue. Plaque assays were performed on the C. glabrata strain CBS138 by applying ergosterol or cholesterol to evaluate the possible reversal of the inhibition induced by compounds 2 , 3 and 4. Inhibition was easily suppressed in all three cases, recovering the viability of C. glabrata. These results reveal that the CgHMGR model is excellent for testing antifungals. Compound 4 produced the best effect and is herein proposed as a new potent antifungal agent. [ABSTRACT FROM AUTHOR]

The emergent pathogen Candida glabrata differs from other yeasts because it assimilates only two sugars, glucose and the disaccharide trehalose. Since rapid identification tests are based on the ability of this yeast to rapidly hydrolyze trehalose, in this work a biochemical and molecular characterization of trehalose catabolism by this yeast was performed. Our results show that C. glabrata consumes and ferments trehalose, with parameters similar to those observed during glucose fermentation. The presence of glucose in the medium during exponential growth on trehalose revealed extracellular hydrolysis of the sugar by a cell surface acid trehalase with a pH optimum of 4.4. Approximately ∼30% of the total enzymatic activity is secreted into the medium during growth on trehalose or glycerol. The secreted enzyme shows an apparent molecular mass of 275 kDa in its native form, but denaturant gel electrophoresis revealed a protein with ∼130 kDa, which due to its migration pattern and strong binding to concanavalin A, indicates that it is probably a dimeric glycoprotein. The secreted acid trehalase shows high affinity and activity for trehalose, with K m and V max values of 3.4 mM and 80 U (mg protein) −1 , respectively. Cloning of the CgATH1 gene ( CAGLOK05137g ) from de C. glabrata genome, a gene showing high homology to fungal acid trehalases, allowed trehalose fermentation after heterologous expression in Saccharomyces cerevisiae. [ABSTRACT FROM AUTHOR]

Only five species account for 92% of cases of candidemia ( Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei); however, their distribution varies in population-based studies conducted in different geographical areas. C. albicans is the most frequent species, but considerable differences are found between the number of cases caused by C. glabrata and C. parapsilosis. Studies from Northern Europe and the USA reported a high number of cases caused by C. glabrata, whereas studies from Spain and Brazil demonstrated a lower number of cases caused by C. glabrata and a higher number of cases attributed to C. parapsilosis. Globally, the frequency of C. albicans is decreasing, while that of C. glabrata and C. krusei is stable, and C. parapsilosis and C. tropicalis are increasing. Patient characteristics and prior antifungal therapy also have a considerable influence on the distribution and frequency of Candida spp., regardless of the geographical area. C. albicans is more frequent in patients aged up to 18 years, the frequency of C. parapsilosis decreases with age, and C. glabrata is more common in the elderly. Finally, the presence of horizontal transmission of Candida spp. isolates (reported mainly in patients from the adult medical and post-surgical ICU, patients from oncology-haematology units, and neonates) can affect species distribution. [ABSTRACT FROM AUTHOR]

Abstract: Since two new species phylogenetically related to Candida glabrata with slightly different phenotypes and antifungal susceptibility profiles have been described, it seems to be necessary from clinical point of view, to develop a rapid and accurate identification system in order to distinguish between these three fungal species. We studied the performance of denaturing high performance liquid chromatography (dHPLC) as a faster (less than 7min) and alternative novel technique for simultaneous analysis of Candida species in different biological matrices. The analyses show the good low limit of detection (LLOD) in all biological matrices studied (5.16–9.56ngμL−1, 4.14–4.70ngμL−1 and 3.99–4.66ngμL−1 for Candida bracarensis, Candida nivariensis and C. glabrata, respectively). 180 Candida isolates were analyzed in order to demonstrate the method suitability for screening analysis to identify C. glabrata and its cryptic species (C. bracarensis and C. nivariensis) in clinical routine. [Copyright &y& Elsevier]

Abstract: Candida glabrata (Cgl) and Candida parapsilosis (Cpa) can cause serious infections and can be resistant to some antifungal drugs. In treating infections caused by these organisms, killing rates and post-antifungal effects (PAFE) are important factors in both dose interval choice and outcome. Two strains each of Cgl and Cpa were studied. For PAFE studies, each organism was exposed to micafungin (MCF), anidulafungin (ANF), or caspofungin (CAS) for 1 h at concentrations ranging from 0.25 to 16×MIC. Cell suspensions were then washed 3 times and resuspended in fresh broth. Time 0 was immediately after resuspension of the yeast. Time-kill experiments were done using similar drug concentrations. Samples were removed at each time point (0-120 h) and viable counts determined. PAFE of ANF and CAS were generally very long, were markedly longer than those of MCF, and increased with increased drug concentration. For ANF and CAS, PAFE for Cgl were greater than those for Cpa only at 0.5 to 2 × MIC. Time-kill experiments showed that ANF, CAS, and MCF were fungicidal at 8 to 16 × MIC up to 120 h. CAS had the greatest activity against Cgl, while ANF and MCF were more active than CAS against Cpa. Because of the prolonged PAFE of these echinocandins, especially ANF and CAS, less frequent dosing during therapy of Cpa and Cgl infections could be considered. Further studies are needed to determine the clinical efficacy of longer dosing intervals. [Copyright &y& Elsevier]

Abstract: Planktonic and attached cells of strains of Candida albicans, C. glabrata and C. krusei with varied susceptibilities to fluconazole (FCZ) were compared for their relative susceptibilities to Ag+ via cell recovery and flow cytometric analyses. All strains lost membrane permeability and were non-recoverable upon culture after 1h exposure in morpholino-ethanesulfonic acid (MES) buffer fortified with ⩽2.0μg/ml Ag+. Cells attached to silicone over a 2-h period demonstrated enhanced tolerance to FCZ and to a lesser degree to Ag+. Minimal inhibitory concentrations of Ag+ in defined media increased in the order C. glabrata, C. krusei, C. albicans. Susceptibilities to Ag+ did not correlate with tolerance or resistance to FCZ. [Copyright &y& Elsevier]

Candidal adherence to mucosal surfaces is considered as the first step in the pathogenesis of oral candidiasis. We examined the effect of antifungal polyenes, amphotericin B, nystatin and natamycin, at sublethal and minimum inhibitory concentrations (MICs) on the adherence of Candida albicans and Candida glabrata to HeLa cervical carcinoma and HSC-3 oral squamous cell carcinoma cells. A total of six oral Candida isolates were used throughout the study. Two Candida strains, C. albicans (44990) and C. glabrata (MYA-275) were obtained from ATCC. Four Candida strains, C. albicans 19 and 24 and C. glabrata 15 and 21, were isolated from patients with documented Candida-associated denture stomatitis. Cells were either incubated with Candida in the presence of the drug, or pre-incubated with yeasts and exposed subsequently to the drug. In the drug-free controls, the mean number of C. albicans yeasts associated with HeLa cells obtained from all experiments (130.1±10.1 yeasts/mm2) was significantly greater than that for HSC-3 cells (114.7±10.1 yeasts/mm2; P<0.025). For C. glabrata, the mean adherence to HeLa and HSC-3 cells was 84.4±5.5 and 84.4±3.3 yeasts/mm2, respectively, and these values were not statistically different (P>0.4). Candidal adherence was significantly reduced when the tested polyenes were present during the “adherence phase”. The obtained values were significantly different from the controls, except for the effect of nystatin at the MIC on the adherence of C. glabrata strain MYA-275 to HeLa cells (P<0.375). Amphotericin B had the highest effect against both Candida species, reducing adherence by ∼50 and ∼60%, at the MIC and sublethal concentrations, respectively. The susceptibility of cell-associated Candida to polyenes was decreased markedly and the treatment did not result in significant detachment of adherent yeasts. The reduction in adherence was between 2 and 10%, when compared to the drug-free controls. These findings suggest that sub-therapeutic levels of polyenes that are likely to persist in the oral cavity following topical treatment may modulate candidal colonization when present during the “adherence phase”. [Copyright &y& Elsevier]

A defined and balanced immunomodulatory response is crucial for the protection of mucosal surfaces being in contact with pathogenic microorganisms. This study examined the local host response mechanisms of epithelial cells in experimental Candida albicans , C. tropicalis , and C. glabrata infections by measuring the expression of cytokines at the mRNA and protein level. During the course of infection with active but not with heat-killed C. albicans stimulation of the gene expression levels for interleukin-1α, interleukin-1β, tumor necrosis factor, Exodus-2, P-selectin ligand, granulocyte-monocyte colony-stimulating factor, and interleukin-8 was observed by standard and quantitative reverse transcription–polymerase chain reaction. This cytokine pattern may favor a chemotactic and a T helper 1 response. Initial moderate or weak upregulation of these cytokine genes by reverse transcription–polymerase chain reaction was also observed in epithelial infection with the less virulent species C. tropicalis and C. glabrata. Heat-killed C. albicans failed to induce an epithelial immune response. At the protein level, expression of interleukin-8 protein was strongly enhanced during the course of C. albicans infection, whereas lower levels were seen with C. tropicalis and C. glabrata . The different expression patterns of cytokines were associated with differences in virulence of the Candida strains. This study's data, therefore, show a correlation between the virulence potential of pathogenic fungi, possibly mediated by specific virulence factors (such as proteinases), and the secretion of epithelial cytokines and chemokines, which may initiate in vivo a protective T helper 1 immunologic response and contribute to the recruitment of activated leukocytes and lymphocytes to the site of mucosal infection. [ABSTRACT FROM AUTHOR]