Do raspberry extracts and fractions have antifungal or anti-adherent potential against Candida spp.?

Candida spp., especially Candida albicans, is one of the main colonisers of the oral cavity. Due to its ability to form biofilms, it can be implicated in dental caries, periodontal disease and denture stomatitis. Microbial cells in biofilms are minimally impacted by conventional drugs. The aim of this study was to find new substances able to inhibit the adhesion of Candida spp. in order to prevent biofilm formation in the oral cavity. This study focused on the red raspberry (Rubus idaeus) fruit, known for its richness in potentially antimicrobial tannins. Extraction with a polarity gradient was performed on acetone extracts from frozen ripe and unripe fruits, resulting in eight extracts. The antifungal and anti-adhesion effects of the extracts were determined using broth microdilution and XTT methods, respectively, against C. albicans, Candida glabrata and Candida parapsilosis strains. Interestingly, four extracts (hexane and ethyl acetate) displayed anti-adhesion activity against C. albicans at low concentrations [50% inhibitory concentration (IC50) 15.6-62.5 µg/mL]. Bioassay-guided fractionation by chromatographic methods of the most active extract obtained from ripe fruit (ethyl acetate extract) led to two subfractions enriched in anti-adhesion compounds, identified by mass spectrometry analysis as hydrolysable and condensed tannins. Their activities were dose-dependent with maximum inhibition at 80% (IC50 = 25 µg/mL and 12.5 µg/mL). Regarding antifungal activity, no extract was active against planktonic cells of the tested strains. This work highlights for the first time the potential of raspberries to prevent oral C. albicans biofilms.

Phytochemical analysis and mode of action against Candida glabrata of Paeonia emodi extracts.

In the present study, we have evaluated the antifungal activity of the seed, root and leaf of Paeonia emodi (commonly known as Himalayan peony) in four common solvents (acetone, chloroform, methanol and water) against six fungal strains. The methanolic seed extract (MSE) showed promising antifungal activity against Candida albicans (6.25mg/mL), Candida glabrata (3.12mg/mL) and Candida parapsilosis (12.50mg/mL) among all the fungal strains tested. Combination of the MSE with the well-known commercial antifungal drugs amphotericin B (Amp B), nystatin (NYS) and fluconazole (FLC) resulted in the killing of C. glabrata at non-inhibitory concentrations, i.e., 0.35µg/mL for Amp B, 0.55µg/mL for NYS and 1.19µg/mL for FLC. Notably, MSE caused cell wall damage of C. glabrata cells, as confirmed by confocal microscopy, flowcytometry and scanning electron microscopy (SEM). The MSE was fractionated by thin layer chromatography (TLC). TLC-bioautography was used to determine the active compounds present in the MSE. Column chromatography was used to separate the potential active compounds from the MSE. Furthermore, gas chromatography-mass spectrometry (GC-MS) andfourier-transform infrared spectroscopy (FTIR) were used to identify the phytocomponents of the MSE. These experiments revealed 13-docosenamide/9-octadecenamide/trans-13-docosenamide (89.70%) as being the predominant compound using a chloroform/methanol solvent system for the separation. Interestingly, the MSE also exhibited less significant cytotoxicity at the minimum inhibitory concentration (MIC) against mammalian cells (HeLa and HEK293). This study suggests that the MSE of P. emodi can be used for the treatment of C. glabrata infection.

Roles of vacuolar H+-ATPase in the oxidative stress response of Candida glabrata.

Vacuolar H(+)-ATPase (V-ATPase) is responsible for the acidification of eukaryotic intracellular compartments and plays an important role in oxidative stress response (OSR), but its molecular bases are largely unknown. Here, we investigated how V-ATPase is involved in the OSR by using a strain lacking VPH2, which encodes an assembly factor of V-ATPase, in the pathogenic fungus Candida glabrata The loss of Vph2 resulted in increased H2O2 sensitivity and intracellular reactive oxygen species (ROS) level independently of mitochondrial functions. The Δvph2 mutant also displayed growth defects under alkaline conditions accompanied by the accumulation of intracellular ROS and these phenotypes were recovered in the presence of the ROS scavenger N-acetyl-l-cysteine. Both expression and activity levels of mitochondrial manganese superoxide dismutase (Sod2) and catalase (Cta1) were decreased in the Δvph2 mutant. Phenotypic analyses of strains lacking and overexpressing these genes revealed that Sod2 and Cta1 play a predominant role in endogenous and exogenous OSR, respectively. Furthermore, supplementation of copper and iron restored the expression of SOD2 specifically in the Δvph2 mutant, suggesting that the homeostasis of intracellular cupper and iron levels maintained by V-ATPase was important for the Sod2-mediated OSR. This report demonstrates novel roles of V-ATPase in the OSR in C. glabrata.

Candida glabrata--unique features and challenges in the clinical management of invasive infections.

Candida glabrata is a pathogenic yeast with several unique biological features. This article provides an up-to-date review on current data and reasoning aspects of this clinically problematic organism. Haploidy, absence of pseudohyphae, facultative anaerobe growth of C. glabrata, as well as its intrinsically low susceptibility to azole antifungals require specific consideration in diagnosis and treatment approaches. As C. glabrata today represents a sizeable percentage of pathogens in candidaemia, the use of azole antifungals in upfront therapy of invasive yeast infections is discouraged by recent guidelines. While the selection of C. glabrata mutants with impaired susceptibility to echinocandins has been described, analyses of several clinical studies indicate an association of improved outcomes with the use of echinocandins as the primary treatment for invasive yeast infections with potential or documented involvement of C. glabrata.

Current recommendations and importance of antifungal stewardship for the management of invasive candidiasis.

Invasive candidiasis can have a major effect on patient prognosis and medical economics. Quickly eliminating the focus of the infection and administering appropriate antifungal therapy are important. Clinical guidelines for invasive candidiasis have been issued in the USA, Europe and recently in Japan. The purpose of this review is to summarize the current recommendations on how to diagnose and treat invasive candidiasis based on the evidence gathered to date and by referencing guidelines from various countries. Echinocandin antifungals play a central role in the prevention and treatment of invasive candidiasis although a recent increase in echinocandin-resistant Candida glabrata is seen as problematic. In the future, promoting the appropriate use of antifungal agents by antifungal stewardship teams will be necessary to suppress adverse effects, appearance of resistant strains and unnecessary medical expenses, as well as improve positive clinical outcomes and prognoses.

[Anti-attachment effect of ethyl acetate extract of Huanglian Jiedu decoction on Candida glabrata].

OBJECTIVE: To investigate anti-attachment effect of ethyl acetate extract of Huanglian Jiedu decoction (EAHD) on Candida glabrata. METHOD: Serial 2-fold dilution assay was used to determine the minimum inhibitory concentrations MICs of EAHD to C. glabrata. XTT assay was used to evaluate the effect of EAHD against adhesion of C. glabrata. Inverted microscope, scanning electron microscope (SEM) and fluorescein diacetate (FDA) staining were applied to observe the morphological changes of C. glabrata in adhesion. PCR was adopted to inspect the expression of attachment-related genes such as EPA1, EPA6 and EPA7. RESULT: The MIC of EAHD and fluconazole to C. glabrata were 320 mg · L(-1) and 1 mg · L(-1) respectively. The total cells including budding cells decreased in a dose-dependent manner following EAHD treatment. The expressions of EPA1, EPA6 and EPA7 were downregulated dramatically after EAHD treatment. CONCLUSION: EAHD could effectively inhibit adherence of C. glabrata.

Comparative effects of hypoxia and hypoxia mimetic cobalt chloride on in vitro adhesion, biofilm formation and susceptibility to amphotericin B of Candida glabrata.

OBJECTIVES: Candida glabrata has emerged as potent pathogen in nosocomial infections. The objective of this study is to investigate the effects of hypoxia (an important host factor) and hypoxia mimetic cobalt chloride upon growth, in vitro adhesion, biofilm formation and susceptibility to amphotericin B of Candida glabrata. MATERIALS AND METHODS: Growth was checked by spotting assays. Expression of TDH3, a gene of glycolytic pathway was analyzed as intracellular hypoxia marker by reverse transcription PCR. In vitro adhesion, biofilm development and susceptibility of biofilm to amphotericin B were performed on polystyrene plates and quantified by XTT assay in RPMI 1640 and YNB media. Experiments were performed in triplicates and Student's t-test was used for statistical analysis. RESULTS: Hypoxia did not compromise the growth of C. glabrata unlike CoCl2. Hypoxia and CoCl2 upregulated TDH3 expression. Adhesion was reduced upon exposure to hypoxia and CoCl2. Biofilm activity remained unchanged in the presence of CoCl2 in both media. In comparison to normoxia control, hypoxia increased biofilm activity to 259.33 ± 22.05% in RPMI 1640, while hypoxia reduced it to 70.99 ± 2.99% in YNB. Biofilm susceptibility to amphotericin B was significantly decreased in RPMI 1640 and remained unaffected in YNB in hypoxia. CONCLUSIONS: C. glabrata grows well even under hypoxia but not upon CoCl2 exposures. CoCl2 mimics hypoxia like expression of TDH3 but affects the virulence properties unlike hypoxia. Both, hypoxia and CoCl2 affects adhesion adversely. Hypoxia increases biofilm development and reduces the susceptibility of biofilm to amp B in RPMI 1640 but not in YNB.

Recurrent arthritis by Candida glabrata, a diagnostic and therapeutic challenge.

Infectious arthritis due to Candida glabrata is very rare. A 40-year-old Iranian man had developed a painful swelling on the left knee since a year ago. A surgery (meniscectomy) was performed on his knee. However, in follow-up visit after 2 months, the patient's condition was deteriorated. Direct examination of synovial fluid with Gram and hematoxylin-eosin stains were negative for any bacterial or fungal infection or crystal elements; however, inoculation into BACTEC™ Mycosis IC/F and Plus Aerobic/F culture bottles led to the isolation of a yeast strain. The macroscopic examination on CHROMagar™ Candida medium combined with microscopical examination on CMT80 agar made a presumptive identification of the isolate to be considered as C. glabrata, and it was later on confirmed by ITS sequencing. Initial empirical treatment was started with intravenous amphotericin B for 4 weeks followed by oral itraconazole which was unsuccessful. Prescription of an oral 150-mg tablet of fluconazole was considered for a 2-month course. All symptoms completely declined, and no recurrence of infection was detected. Antifungal susceptibility testing (AFST) was performed for this isolate, and the result showed sensitivity to both amphotericin B and itraconazole and less susceptibility to fluconazole while clinical recovery was achieved by fluconazole. In any suspected clinical case caused by infectious agents, application of an effective fungal diagnostic test should be considered to avoid complications due to misdiagnosis. The correlation of AFST result with real in vivo therapeutic responses can be strain or patient dependent, and this should be considered for a successive treatment.

Antibiotic exposure as a risk factor for fluconazole-resistant Candida bloodstream infection.

Recent exposure to azoles is an important risk factor for infection with fluconazole-resistant Candida spp., but little is known about the role of antibacterial drug exposure in the emergence of drug-resistant Candida. We did a prospective nationwide surveillance study of candidemia in Israel and analyzed the propensity score-adjusted association between antifungal and antibacterial drug exposure and bloodstream infection with C. glabrata and fluconazole-resistant Candida isolates. Four hundred forty-four episodes of candidemia (450 Candida isolates, 69 [15%] C. glabrata isolates, and 38 [8.5%] fluconazole-resistant isolates) from 18 medical centers in Israel were included. C. glabrata bloodstream infection was strongly associated with recent metronidazole exposure (odds ratio [OR], 3.2; P < 0.001). Infection with a fluconazole-resistant isolate was associated with exposure to carbapenems, trimethoprim-sulfamethoxazole, clindamycin, and colistin (odds ratio, 2.8; P = 0.01). The inclusion of antibacterial drug exposure in a multivariable model significantly enhanced the model's predictive accuracy for fluconazole-resistant Candida bloodstream infection. Our findings may be relevant to the selection of empirical antifungal treatment and broaden the scope of antibiotic-associated collateral damage.

Fungicidal effect of photodynamic therapy against fluconazole-resistant Candida albicans and Candida glabrata.

Although photodynamic therapy (PDT) has shown great promise for the inactivation of Candida species, its effectiveness against azole-resistant pathogens remains poorly documented. This in vitro study describes the association of Photogem® (Photogem, Moscow, Russia) with LED (light emitting diode) light for the photoinactivation of fluconazole-resistant (FR) and American Type Culture Collection (ATCC) strains of Candida albicans and Candida glabrata. Suspensions of each Candida strain were treated with five Photogem® concentrations and exposed to four LED light fluences (14, 24, 34 or 50 min of illumination). After incubation (48 h at 37 °C), colonies were counted (CFU ml(-1)). Single-species biofilms were generated on cellulose membrane filters, treated with 25.0 mg l(-1) of Photogem® and illuminated at 37.5 J cm(-2). The biofilms were then disrupted and the viable yeast cells present were determined. Planktonic suspensions of FR strains were effectively killed after PDT. It was observed that the fungicidal effect of PDT was strain-dependent. Significant decreases in biofilm viability were observed for three strains of C. albicans and for two strains of C. glabrata. The results of this investigation demonstrated that although PDT was effective against Candida species, fluconazole-resistant strains showed reduced sensitivity to PDT. Moreover, single-species biofilms were less susceptible to PDT than their planktonic counterparts.

In vitro evaluation of antibiotic lock technique for the treatment of Candida albicans, C. glabrata, and C. tropicalis biofilms.

Candidaemia associated with intravascular catheter-associated infections is of great concern due to the resulting high morbidity and mortality. The antibiotic lock technique (ALT) was previously introduced to treat catheter-associated bacterial infections without removal of catheter. So far, the efficacy of ALT against Candida infections has not been rigorously evaluated. We investigated in vitro activity of ALT against Candida biofilms formed by C. albicans, C. glabrata, and C. tropicalis using five antifungal agents (caspofungin, amphotericin B, itraconazole, fluconazole, and voriconazole). The effectiveness of antifungal treatment was assayed by monitoring viable cell counts after exposure to 1 mg/mL solutions of each antibiotic. Fluconazole, itraconazole, and voriconazole eliminated detectable viability in the biofilms of all Candida species within 7, 10, and 14 days, respectively, while caspofungin and amphotericin B did not completely kill fungi in C. albicans and C. glabrata biofilms within 14 days. For C. tropicalis biofilm, caspofungin lock achieved eradication more rapidly than amphotericin B and three azoles. Our study suggests that azoles may be useful ALT agents in the treatment of catheter-related candidemia.

Breakthrough fungal infections in stem cell transplant recipients receiving voriconazole.

Infection with voriconazole-resistant fungi may become problematic, because organisms with decreased susceptibility have been noted. Breakthrough fungal infections occurred in 13 of 139 patients who received voriconazole at our center during the period of September 1998 through September 2003. Zygomycetes were found in 6 patients, and Candida glabrata bloodstream infection occurred in 4 patients. Minimal inhibitory concentrations were > or =1 microg/mL for all available isolates. Yeasts and molds with decreased susceptibility to voriconazole may cause invasive infection in patients treated successfully for aspergillosis.