New chromogenic agar medium for the identification of Candida spp

A new chromogenic agar medium (Candida diagnostic agar [CDA]) for differentiation of Candida spp. is described. This medium is based on Sabouraud dextrose agar (Oxoid CM41) and contains (per liter) 40.0 g of glucose, 10.0 g of mycological peptone, and 15.0 g of agar along with a novel chromogenic glucosaminidase substrate, ammonium 4-(2-[4-(2-acetamido-2-deoxy-beta-D-glucopyranosyloxy)-3-methoxyphenyl]-vinyl)-1-(propan-3-yl-oate)-quinolium bromide (0.32 g liter(-1)). The glucosaminidase substrate in CDA was hydrolyzed by Candida albicans and Candida dubliniensis, yielding white colonies with deep-red spots on a yellow transparent background after 24 to 48 h of incubation at 37 degrees C. Colonies of Candida tropicalis and Candida kefyr were uniformly pink, and colonies of other Candida spp., including Candida glabrata and Candida parapsilosis, were white. CDA was evaluated by using 115 test strains of Candida spp. and other clinically important yeasts and was compared with two commercially available chromogenic agars (Candida ID agar [bioMerieux] and CHROMagar Candida [CHROMagar Company Ltd.]). On all three agars, colonies of C. albicans were not distinguished from colonies of C. dubliniensis. However, for the group containing C. albicans plus C. dubliniensis, both the sensitivity and the specificity of detection when CDA was used were 100%, compared with values of 97.6 and 100%, respectively, with CHROMagar Candida and 100 and 96.8%, respectively, with Candida ID agar. In addition, for the group containing C. tropicalis plus C. kefyr, the sensitivity and specificity of detection when CDA was used were also 100%, compared with 72.7 and 98.1%, respectively, with CHROMagar Candida. Candida ID agar did not differentiate C. tropicalis and C. kefyr strains but did differentiate members of a broader group (C. tropicalis, C. kefyr, Candida lusitaniae plus Candida guilliermondii); the sensitivity and specificity of detection for members of this group were 94.7 and 93.8%, respectively. In addition to the increased sensitivity and/or specificity of Candida detection when CDA was used, differentiation of colony types on CDA (red spotted, pink, or no color) was unambiguous and did not require precise assessment of colony color.     

The genusCandida berkhout

The authors attempted to classify a group of five strains excluded in typing of the speciesCandida albicans (Robin) Berkhout because they displayed a relationship toCandida tropicalis (Cast.) Berkhout. They were found to include transitional forms showing progressive development to a higher type. Strain 29-3-32 formed a lower stage of transition fromCandida albicans toCandida tropicalis and was more similar toCandida albicans. Strain 29-3-58 formed a higher transitional stage and was more similar toCandida tropicalis. Strain 29-3-5 was similar toCandida albicans and formed the transition from the latter to strain 29-3-100, which was closely related toCandida intermedia andCandida tropicalis. Strain 29-3-68 formed the transition fromCandida guilliermondii toCandida intermedia and was similar toCandida guilliermondii and the related speciesCandida melibiosi.     

Candida glabrata and Candida albicans; dissimilar tissue tropism and infectivity in a gnotobiotic model of mucosal candidiasis

Germ-free transgenic epsilon 26 (Tgepsilon26) mice, deficient in both natural killer (NK)- and T-cells, were inoculated (orally) with each of two Candida glabrata (BG2 or BG1003) or Candida albicans (CAF2-1 or SC5314) strains. Candida glabrata- or C. albicans-colonized mice exhibited similar numbers of viable Candida in the alimentary tract. Neither C. glabrata nor C. albicans caused systemic candidiasis of endogenous (alimentary tract) origin. Candida albicans invaded oroesophageal (tongue, palate, esophagus) and keratinized gastric tissues, evoked hyperkeratosis and a prominent, chronic, granulocyte-dominated, inflammatory response in all infected tissues, stimulated the production of splenic granulocytes and was lethal for the mice within 3-5 weeks after oral colonization. The two C. glabrata strains colonized the alimentary tract and penetrated into the keratinized (cardia-antrum) gastric tissues, but in contrast to C. albicans, were unable to infect oroesophageal tissues. Furthermore, C. glabrata strains were not lethal for the Tgepsilon26 mice, and did not evoke an inflammatory response in colonized gastric tissues or stimulate the production of splenic granulocytes. This 'stealth-like' behavior could explain the ability of C. glabrata to persist in infected tissues and survive as a commensal in the alimentary tract.     

Comparative evaluation of ATB Fungus 2 and Sensititre YeastOne panels for testing in vitro Candida antifungal susceptibility

ATB Fungus 2 and SensititreYeastOne are commercial methods for antifungal susceptibility testing of yeasts. The agreement between these two methods was assessed with a total of 133 Candida strains (60 Candida albicans, 18 Candida dubliniensis, 29 Candida glabrata, and 26 Candida krusei). MIC endpoints were established after 24 h of incubation at 36-/+1 degrees C by each method. Intra-laboratory reproducibility of both methods was excellent (=or>99%). Overall agreement between ATB Fungus 2 and Sensititre YeastOne 3 MICs (within 2 dilutions) was 91.2-97.7% for amphotericin B, 5-fluorocytosine and itraconazole, and 82.7% for fluconazole. The categorical agreement when ATB Fungus 2 results were compared to those by SensititreYeastOne 3 was 93.2-98.5% for 5-fluorocytosine and amphotericin B, but lower for the triazoles (72.9-75.9%). This easy to perform method could be an alternative for routine use in the clinical microbiology laboratory for susceptibility testing of common Candida spp.     

Distribution of a corticosteroid-binding protein in Candida and other fungal genera

Using [3H]corticosterone as a probe, corticosteroid-binding protein (CBP) was detected in eight out of eight isolates of Candida albicans, of both A and B serotypes. The apparent dissociation constant (Kd) in the various isolates ranged between 8 and 19 nM; the binding capacity varied from 122 to over 2400 fmol (mg cytosol protein)-1. There was no correlation between the amount or affinity of CBP and isolate virulence for murine hosts. Further analysis revealed demonstrable CBP in six out of six Candida species other than C. albicans. One isolate of C. tropicalis has been identified which fails to bind [3H]corticosterone. Saccharomyces cerevisiae, Neurospora crassa and Paracoccidioides brasiliensis also failed to bind [3H]corticosterone. Preliminary attempts were made to determine functions mediated by CBP in Candida, but in vitro growth, phase conversion and glucose oxidation by Candida were unaffected by the addition of a variety of steroid hormones. These data indicate that the presence of CBP in Candida does not correlate with either virulence or serotype. The physiological significance of CBP remains to be determined.     

Amylolytic activity of yeasts

The total amylolytic activity of 14 selected fermentation type I members of the generaSaccharomyces, Zygosaccharomyces, Candida andTorulopsis was studied. Fractions with α-glucosidase activity, the specificty of which was tested for maltose and sucrose, were isolated on carboxymethyl-cellulose from the intracellular contents of two strains ofCandida albicans and one ofCandida stellatoidea. The fraction from the strainCandida albicans 29-3-109, which is more virulent for mice, displayed the greatest α-glucosidase activity, moderate activity was present in the strainCandida stellatoidea 29-64-1, while the lowest activity was found in the less virulent strain ofCandida albicans, 29-3-19.     

Antimicrobial and anti-lipase activity of quercetin and its C2-C16 3-O-acyl-esters.

Neither quercetin (Q), nor 3-O-acylquercetines, up to 100 microg/mL, had any significant activity on selected gram-positive strains (Staphylococcus aureus, Bacillus subtilis, Listeria ivanovi, Listeria monocytogenes, Listeria serligeri), gram-negative strains (Escherichia coli, Shigella flexneri, Shigella sonnei, Salmonella enteritidis, Salmonella tiphymurium) and yeasts (Candida albicans and Candida glabrata). In addition, we confirmed the known anti-HIV activity of Q (80% inhibition at 40 microM), which might depend on the free hydroxyl in the C-3 position, as suggested by the lack of activity of the 3-O-acylquercetines. Finally, we described an interesting inhibitory activity on Candida rugosa lipase by Q (IC(16)=10(-4) M) and its esters (3-O-acylquercetines) which, in vivo, could play an important role against lipase producing microorganisms. In particular, 3-O-acyl-quercetines, being more active (IC(16)=10(-4)-10(-6) M) and more lipophilic, could be more effective than Q when applied to the skin or mucosae, and deserve to be studied further.     

Antifungal susceptibility of epigallocatechin 3-O-gallate (EGCg) on clinical isolates of pathogenic yeasts

This is the first report to investigate the antifungal susceptibility of 21 clinical isolates of seven Candida species to epigallocatechin 3-O-gallate (EGCg) and to compare with six antifungal agents, amphotericin B (AMPH), fluconazole (FLCZ), flucytosin (5FC), itraconazole (ITCZ), micafungin (MCFG), and miconazole (MCZ), using a method following the National Committee for Clinical Laboratory Standards (NCCLS) M27-A guidelines. Among the tested species, Candida glabrata exhibited the highest susceptibility to EGCg (MIC50, 0.5-1 microg/ml and MIC90, 1-2 microg/ml) compared favorably with FLCZ, although they were slightly less susceptible than to AMPH, 5FC, MCFG, ITCZ, and MCZ. Candida guilliemondii and Candida parapsilosis (MIC50, 1-4 microg/ml and MIC90, 2-16 microg/ml) were also susceptible to EGCg, although they appear to be slightly less susceptible to EGCg than C. glabrata and the other antifungal agents tested. Moreover, the susceptibility of Candida krusei strains (MIC50, 2 microg/ml and MIC90, 4-8 microg/ml) to EGCg was approximately 2- to 8-fold higher than those of 5FC and FLCZ. Our data indicate that EGCg can inhibit clinically pathogenic Candida species, although the concentrations of EGCg for antifungal susceptibility were slightly higher than those of tested antifungal agents on the whole. Based on these results, we suggest that EGCg may be effectively used as a possible agent or adjuvant for antifungal therapy in Candidiasis.     

Characterization of two different toxins of Wickerhamomyces anomalus (Pichia anomala) VKM Y-159

Wickerhamomyces anomalus VKM Y-159 strain produces two types of toxin designated as WAKT a and WAKT b, encoded by chromosomal genes. The WAKT a toxin is heat-labile, pronase sensitive acting in pH range 3-4 affecting on several yeasts including pathogenic Candida species while the WAKT b toxin is protease- and thermo-resistant, acting in pH range 3-7 on two species, Candida alai and Candida norvegica. The rapid decrease of the number of viable cells after toxin treatment demonstrates that both toxins have cytocidic effect.     

Tissue-specific human beta-defensins (HBD)-1, HBD-2 and HBD-3 secretion profile from human amniochorionic membranes stimulated with candida albicans in a two-compartment tissue culture system

ABSTRACT: BACKGROUND: During intrauterine infection, amniochorionic membranes represent a mechanical and immunological barrier against dissemination of infection. Human beta defensins (HBD)-1, HBD-2, and HBD-3 are key elements of innate immunity that represent the first line of defense against different pathogen microorganisms associated with preterm labor. The aim of this work was to characterize the individual contribution of the amnion (AMN) and choriodecidua (CHD) regions to the secretion of HBD-1, HBD-2 and HBD-3, after stimulation with Candida albicans. METHODS: Full-thickness human amniochorionic membranes were obtained after delivery by elective cesarean section from women at 37-40 wk of gestation with no evidence of active labor. The membranes were cultured in a two-compartment experimental model in which the upper compartment is delimited by the amnion and the lower chamber by the choriodecidual membrane. One million of Candida albicans were added to either the AMN or the CHD face or to both and compartmentalized secretion profiles of HBD-1, HBD-2, and HBD-3 were quantified by ELISA. Tissue immunolocalization was performed to detect the presence of HBD-1, -2, -3 in tissue sections stimulated with Candida albicans. RESULTS: HBD-1 secretion level by the CHD compartment increased 2.6 times (27.30 [20.9-38.25] pg/micrograms protein) when the stimulus with Candida albicans was applied only on this side of the membrane and 2.4 times (26.55 [19.4-42.5] pg/micrograms protein) when applied to both compartments simultaneously. HBD-1 in the amniotic compartment remained without significant changes.HBD-2 secretion level increased significantly in the CHD when the stimulus was applied only to this region (2.49 [1.49-2.95] pg/micrograms protein) and simultaneously to both compartments (2.14 [1.67- 2.91] pg/micrograms protein). When the stimulus was done in the amniotic compartment HBD-2 remained without significant changes in both compartments.HBD-3 remained without significant changes in both compartments regardless of the stimulation modality. Localization of immune-reactive forms of HBD-1, HBD-2, and HBD-3 was carried out by immunohistochemistry confirming the cellular origin of these peptides. CONCLUSION: Selective stimulation of amniochorionic membranes with Candida albicans resulted in tissue-specific secretion of HBD-1 and HBD-2, mainly in the CHD, which is the first region to become infected during an ascending infection.     

THIS ARTICLE HAS BEEN RETRACTEDAerosolised liposomal hamycin for treatment of systemic Candida infections in mice

Mice lethally infected with Candida albicans were exposed to small-particle aerosols containing hamycin liposomes. The drug, when administered twice daily for 2 h (0.68 mg kg(-1) body weight per day) on days 1, 2 and 3 post inoculation, significantly reduced the numbers of Candida organisms in the kidneys. Aerosol treatment increased the survival time of mice given two 2-h treatments once a week for 4 weeks. A twice weekly 2-h small particle aerosol administration of hamycin for 1, 2, or 3 weeks significantly increased both the mean time of survival and percent survival.     

Metal ions may suppress or enhance cellular differentiation in Candida albicans and Candida tropicalis biofilms

Candida albicans and Candida tropicalis are polymorphic fungi that develop antimicrobial-resistant biofilm communities that are characterized by multiple cell morphotypes. This study investigated cell type interconversion and drug and metal resistance as well as community organization in biofilms of these microorganisms that were exposed to metal ions. To study this, Candida biofilms were grown either in microtiter plates containing gradient arrays of metal ions or in the Calgary Biofilm Device for high-throughput susceptibility testing. Biofilm formation and antifungal resistance were evaluated by viable cell counts, tetrazolium salt reduction, light microscopy, and confocal laser scanning microscopy in conjunction with three-dimensional visualization. We discovered that subinhibitory concentrations of certain metal ions (CrO(4)(2-), Co(2+), Cu(2+), Ag(+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), AsO(2)(-), and SeO(3)(2-)) caused changes in biofilm structure by blocking or eliciting the transition between yeast and hyphal cell types. Four distinct biofilm community structure types were discerned from these data, which were designated "domed," "layer cake," "flat," and "mycelial." This study suggests that Candida biofilm populations may respond to metal ions to form cell-cell and solid-surface-attached assemblages with distinct patterns of cellular differentiation.     

A novel bovine lactoferrin peptide, FKCRRWQWRM, suppresses Candida cell growth and activates neutrophils.

To identify potent new antifungal agents, the Candida cell growth inhibitory activities of six lactoferrin (Lf) peptides consisting of 6-25 amino acid residues (peptide 1, FKCRRWQWRMKKLGAPSITCVRRAF lactoferricin B; peptide 2, FKCRRWQWRM; peptide 2', FKARRWQWRM; peptide 3, GAPSITCVRRAF; peptide 4, RRWQWR; and peptide 5, RWQWRM) were examined. Of these, peptide 2 strongly suppressed the multiplication of Candida cells, but other peptides showed only weak activities. In two strains of C. albicans, the minimum inhibitory concentration 100 of peptide 2 (17.3+/-2.2 microM and 17.5+/-2.4 microM) was close to that of miconazole (13.0+/-1.7 microM and 13.1+/-1.6 microM) but markedly different from that of amphotericin B (0.52+/-0.09 microM and 0.56+/-0.11 microM). The suppression of Candida cell growth was additively increased by a combination of peptide 2 with amphotericin B and miconazole. Peptides 1, 3, 4 and 5 and Lf suppressed iron uptake by Candida cells, inversely correlated with their Candida cell growth inhibition activities. However, iron uptake was not inhibited by peptide 2. In addition, peptide 2 upregulated Candida cell killing activity of polymorphonuclear leukocytes (PMN) increasing their superoxide generation, protein kinase C activity, p38 MAPK activity and the expression of p47phox. These results indicated that the main antimicrobial activity of the Lf peptides is dependent on the N-terminal half of Lf and that the PMN upregulatory activity of peptide 2 and additive function of peptide 2 with antifungal drugs are useful for prophylaxis and control of candidiasis.     

Herpes simplex virus-1 entrapped in <Emphasis Type="Italic">Candida albicans</Emphasis> biofilm displays decreased sensitivity to antivirals and UVA1 laser treatment

Background

Recently, we published data suggesting a mutualistic relationship between HSV-1 and Candida. albicans; in particular: (a) HSV-1 infected macrophages are inhibited in their anti-Candida effector function and (b) Candida biofilm protects HSV-1 from inactivation. The present in vitro study is aimed at testing the effects of Candida biofilm on HSV-1 sensitivity to pharmacological and physical stress, such as antiviral drugs (acyclovir and foscarnet) and laser UVA1 irradiation. We also investigated whether fungus growth pattern, either sessile or planktonic, influences HSV-1 sensitivity to antivirals.

Methods

Mature Candida biofilms were exposed to HSV-1 and then irradiated with laser light (UVA1, 355 λ). In another set of experiments, mature Candida biofilm were co-cultured with HSV-1 infected VERO cells in the presence of different concentrations of acyclovir or foscarnet. In both protocols, controls unexposed to laser or drugs were included. The viral yield of treated and untreated samples was evaluated by end-point titration. To evaluate whether this protective effect might occur in relation with a different growth pattern, HSV-1 infected cells were co-cultured with either sessile or planktonic forms of Candida and then assessed for susceptibility to antiviral drugs.

Results

UVA1 irradiation caused a 2 Log reduction of virus yield in the control cultures whereas the reduction was only 1 Log with Candida biofilm, regardless to the laser dose applied to the experimental samples (50 or 100 J/cm²). The presence of biofilm increased the IC₉₀ from 18.4–25.6 J/cm². Acyclovir caused a 2.3 Log reduction of virus yield in the control cultures whereas with Candida biofilm the reduction was only 0.5 Log; foscarnet determined a reduction of 1.4 Log in the controls and 0.2 Log in biofilm cultures. Consequently, the ICs₅₀ for acyclovir and foscarnet increased by 4- and 12-folds, respectively, compared to controls. When HSV-1 was exposed to either sessile or planktonic fungal cells, the antiviral treatments caused approximately the same weak reduction of virus yield.

Conclusions

These data demonstrate that: (1) HSV-1 encompassed in Candida biofilm is protected from inactivation by physical (laser) and pharmacological (acyclovir or foscarnet) treatments; (2) the drug antiviral activity is reduced at a similar extent for both sessile or planktonic Candida.

     

焦性没食子酸联合唑类药物对念珠菌的抑菌作用

目的了解光滑念珠菌临床菌株的药敏情况以及中药单体焦性没食子酸联合唑类药物对念珠菌的抑菌作用。方法收集临床分离的光滑念珠菌116株、白念珠菌49株、热带念珠菌42株、克柔念珠菌4株和近平滑念珠菌13株,采用ATB FUNGUS3药敏试条检测光滑念珠菌的药敏情况;同时采用棋盘肉汤稀释法检测焦性没食子酸联合唑类药物对念珠菌的抑菌情况。结果116株光滑念珠菌中,14.66%(17株)的菌株对氟康唑耐药,22.41%(26株)对伊曲康唑表现为非野生型和81.03%(94株)对伏立康唑表现为非野生型。焦性没食子酸对5种念珠菌的抑菌情况,46.55%光滑念珠菌的MIC值为64μg/mL;34.69%白念珠菌的MIC值为64μg/mL;59.52%热带念珠菌的MIC值为64μg/mL;25%克柔念珠菌的MIC值为128μg/mL;46.15%近平滑念珠菌的MIC值为128μg/mL。唑类药物与焦性没食子酸联合用药时,100%、99.14%、99.14%的光滑念珠菌分别对氟康唑、伊曲康唑和伏立康唑表现为协同作用或相加作用,差异无统计学意义(P>0.05);而白念珠菌、热带念珠菌、克柔念珠菌和近平滑念珠菌均表现为无关作用和拮抗作用。与单独用药相比,联合用药时81.03%、68.1%、77.59%的光滑念珠菌分别对氟康唑、伊曲康唑、伏立康唑的MIC值降低2~3个浓度梯度,且耐药组与非耐药组之间差异具有统计学意义(P<0.05)。结论光滑念珠菌对唑类药物具有耐药性,伏立康唑非野生型菌株所占比例最高。焦性没食子酸单独用药时,5组念珠菌中对光滑念珠菌的抑菌效果最好,且敏感组比耐药组的抑菌效果更加显著。焦性没食子酸联合唑类药物显著降低了光滑念珠菌唑类药物的MIC值,且耐药组比非耐药组的效果更加显著,为中西医结合治疗临床光滑念珠菌感染提供实验依据。     

The acetates of p-nitrophenyl alpha-L-arabinofuranoside--regioselective preparation by action of lipases

All possible di-O-acetates and mono-O-acetates of p-nitrophenyl alpha-L-arabinofuranoside were prepared by chemoenzymatic way using lipases. The 2,3-di-O-acetate was obtained in 90% yield by deacetylation of the primary acetyl group of per-O-acetylated p-nitrophenyl alpha-L-arabinofuranoside by Candida cylindracea lipase (CCL) or Candida rugosa lipase (LAY). The 2,5- and 3,5-di-O-acetates were obtained by acetylation of p-nitrophenyl alpha-L-arabinofuranoside by Pseudomonas cepacia lipase (LPS-30) in organic solvents. The 5-O-acetate was regioselectively synthesised in 95% yield by acetylation of p-nitrophenyl alpha-L-arabinofuranoside catalysed by porcine pancreas lipase. Finally, the 2- and 3-O-acetates of p-nitrophenyl alpha-L-arabinofuranoside were obtained in two steps. The enzymatic di-O-acetylation of p-nitrophenyl alpha-L-arabinofuranoside by LPS-30 was followed by enzymatic hydrolysis of the primary acetyl group by CCL or LAY.     

Saliva promotes survival and even proliferation of Candida species in tap water

Candida yeasts colonize the human oral cavity as commensals or opportunistic pathogens. They may be isolated from water circulating in dental unit waterlines mixed with traces of saliva mainly because of the dysfunction of antiretraction valves. This study deals with the growth ability of Candida albicans, Candida glabrata and Candida parapsilosis in tap water with saliva (0-20% v/v). Results show that C. glabrata is the most susceptible species in tap water. Furthermore, saliva promotes both survival and proliferation of the three studied Candida species in tap water.     

Synthesis and antifungal activity of 5-arylamino-4,7-dioxobenzo[b]thiophenes

5-Arylamino-4,7-dioxobenzo[b]thiophenes 3-6 were synthesized and tested for in vitro antifungal activity against Candida and Aspergillus species. 5-Arylamino-6-chloro-2-(methoxycarbonyl)-4,7-dioxobenzo[b]thiophenes 5 showed, in general, more potent antifungal activity against Candida species than the other 4,7-dioxobenzo[b]thiophenes 3, 4 and 6. The results suggest that 5-arylamino-4,7-dioxobenzo[b]thiophenes would be potent antifungal agents.     

Synthesis and SAR studies of biaryloxy-substituted triazoles as antifungal agents

A series of 1-(substituted biaryloxy)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl) propan-2-ol were synthesized and their antifungal activities were evaluated against eight human pathogenic fungi in vitro. Seventeen compounds showed activity 4- to 64-fold higher than voriconazole against Candida albicans. SAR clearly suggested that introduction of a biaryloxy side chain greatly enhanced the antifungal activity of triazole analogs against Candida species.