Turbidometric characterization of the postantifungal effect: comparative studies with amphotericin B, 5-fluorocytosine and miconazole on Candida albicans

The phenomenon of persistent suppression of Candida albicans yeast cell growth after short drug exposures (postantifungal effect, PAFE) was determined by performing comparative studies using different concentrations of 5-fluorocytosine (5-FC), amphotericin B (AMPH) and miconazole (MCZ). An in vitro turbidometric method was used to measure cell growth and to quantitate the PAFE after removal of the drug by dilution following exposure to C. albicans yeast cells for 0.5 h, 1 h or 2 h. The PAFE was determined by the difference in time (h) required for growth of the control and test cultures to increase to the 0.5 absorbance level following removal of the antifungal agent. A PAFE was demonstrated with each agent and generally the length of the PAFE was dependent upon the concentration of the drug and the time of exposure. An exposure time of 0.5 h resulted in PAFEs ranging from 0.6 to 16.7 h with 5-FC, 0 to 16.5 h with AMPH and 0.1 to 14.1 h with MCZ. In most instances exposure of the cells to each drug for 1 or 2 h resulted in slightly longer PAFEs, respectively. Longer PAFEs were induced with lower concentrations of 5-FC as compared to AMPH and MCZ. The data from such PAFE assays may be useful for determining in vivo treatment regimens, since longer PAFEs may allow for intermittent dosing instead of continuous drug administration.     

Reassessment of the in vitro synergistic effect of fluconazole with the non-steroidal anti-inflammatory agent ibuprofen against Candida albicans

We reassessed the in vitro synergistic effect of fluconazole with the non-steroidal anti-inflammatory agent ibuprofen against the pathogenic yeast Candida albicans. No synergistic effect of fluconazole combined with ibuprofen was seen against fluconazole-susceptible strains, but a remarkable effect was seen against fluconazole-resistant strains (FIX index: 0.02-0.03). Furthermore, vigorous growth of the microorganism, the so-called 'Eagle effect', was observed at concentrations higher than the minimal inhibitory concentrations of ibuprofen and fluconazole. Our results suggest that the combination of ibuprofen and fluconazole should prove useful for treating infection caused by fluconazole-resistant C. albicans.     

In vitro synergy of pseudolaric acid B and fluconazole against clinical isolates of Candida albicans

Candida albicans is the most common fungal pathogen in humans. The emergence of resistance to azole antifungals has raised the issue of using such antifungals in combination to optimise therapeutic outcome. The objective of this study was to evaluate in vitro synergy of pseudolaric acid B (PAB) and fluconazole (FLC) against clinical isolates of C. albicans. The in vitro antifungal activity of PAB, a diterpene acid from Pseudolarix kaempferi Gordon, was evaluated alone and in combination with FLC against 22 FLC-resistant (FLC-R) and 12 FLC-susceptible (FLC-S) C. albicans using the chequerboard microdilution method and time-killing test assays. Synergism was observed in all 22 (100%) FLC-R strains tested as determined by both fractional inhibitory concentration index (FICI) with values ranging from 0.02 to 0.13 and bliss independence (BI) models. Synergism was observed in two of 12 (17%) FLC-S strains as determined by FICI model with values ranging from 0.25 to 0.5 and in three of 12 (18%) FLC-S strains as determined by BI model. For FLC-R strains, the drug concentrations of FLC and PAB, where synergistic interactions were found, ranged from 0.06 to 4 μg ml(-1) and 0.5 to 4 μg ml(-1) respectively. For FLC-S strains, the drug concentrations of FLC and PAB were 1-8 μg ml(-1) and 0.5-4 μg ml(-1) respectively. The BI model gave results consistent with FICI, but no antagonistic activity was observed in any of the strains tested. These interactions between PAB and FLC were confirmed using the time-killing test for the selected strains. Fluconazole and PAB exhibited a good synergism against azole-R isolates of C. albicans.     

Influence of voriconazole and fluconazole on Candida albicans in long-time continuous flow culture

We investigated the influence of voriconazole and fluconazole in a long term trial of continuous flow culture (cfc) up to 9 days. The effects of these azoles were different in dependence on the growth circumstances. Under anaerobic conditions a fungicidal effect of voriconazole was detectable, defined by an inhibition of 99.9%. This also applied to fluconazole for the majority of tested strains of C. albicans. Under aerobic conditions with an otherwise similar situation we found only a fungistatic reaction (inhibition of 90%). Fluorescence microscopy comparing fungal morphology in biofilms on glass surfaces in the cfc revealed a differentiation into blastospores, germ tubes, pseudomycelia and mycelia in the control trial after a cultivation of 8 days. Under anaerobic conditions with azoles only some single cells could be found, sometimes in cell detritus. The adhesion was clearly reduced. Under aerobic conditions more blastospores but no differentiated mycelia were to be seen.     

Interactions between amphotericin B and nitroimidazoles against Candida albicans

Summary. This work proved that nitroimidazole antiprotozoal agents, such as metronidazole, ornidazole, secnidazole and tinidazole, in concentrations of up to 64 μg ml^-1 did not present any antifungal activity against 17 strains of Candida albicans. The combination of each drug with amphotericin B showed the occurrence of variable interactions according to the studied strain. Promising results were observed based on synergistic and additive interactions of the polyene with the metronidazole; the inhibitory and lethal activities of the drugs were potentiated against all strains in concentrations reachable in vivo.
Zusammenfassung. In der vorliegenden Studie konnte gezeigt werden, daß die Nitroimidazol-Antiparasitika Metronidazol, Ornidazol, Secnidazol und Tinidazol in Konzentrationen bis zu 64 μg ml^-1 allein keine antimyzetische Aktivität gegen 17 C. albicans -Stämme aufwiesen. Die Anwendung dieser Agenzien gemeinsam mit Amphotericin B zeigte unterschiedliche Wirkungen auf die untersuchten Stämme. Erfolgversprechende Ergebnisse wurden als synergistische und additive Effekte bei der Kombination der einzelnen Nitroimidazole mit Amphotericin B beobachtet. Konzentrationen, die in vivo anwendbar sind, potenzierten die inhibitorischen und letalen Aktivitäten der Agenzien gegen alle untersuchten C. albicans -Stämme.     

Activity of terbinafine against serious fungal pathogens

Although primarily indicated for dermatophyte infections, the allylamine terbinafine is active in vitro against a broad spectrum of filamentous and dimorphic fungi, in most cases with a primary fungicidal action. Using the standard NCCLS M27-A assay, recent studies confirmed the high activity of terbinafine against dematiaceous fungi and other medically important moulds such as Aspergillus and Penicillium marneffei. Terbinafine displayed a geometric mean MIC of 1.4 micrograms/ml against Candida albicans (n = 259) and has significant in vitro activity against other species of Candida, Cryptococcus, Trichosporon and Blastoschizomyces. As an approach to treatment of refractory infections, interactions of terbinafine with azoles and other agents are being investigated. Terbinafine was synergistic with azoles (and in some cases amphotericin B) against Candida species, Trichosporon beigelii, Aspergillus species, Pseudallescheria boydii and Scopulariopsis brevicaulis, some of which were unresponsive to any drug used singly. Terbinafine combined with fluconazole showed potent synergy against fluconazole- and multidrug-resistant C. albicans isolates. In conclusion, recent in vitro data suggest that terbinafine, either alone or in combination with other antifungal drugs, has potential in the therapy of a range of more severe fungal infections, in addition to its current widespread use against dermatomycoses.     

Susceptibility and trailing growth of Candida albicans to fluconazole: results of a Korean multicentre study

The work reported here is the first nationwide, multicenter surveillance study conducted in Korea to obtain data on fluconazole susceptibility of Candida albicans (C. albicans) isolates. A total of 1137 isolates of C. albicans obtained from 17 university hospitals in South Korea during the 6-month period, July through December 2004, were tested. No resistant strains were observed in any of the isolates. Only five of the 1137 isolates (0.44%) of C. albicans were found to be susceptible dose dependent, with all remaining strains (99.56%) susceptible to fluconazole. Trailing growth at 48 h was found in only four isolates (0.35%).     

Correlation between phospholipase of Candida albicans and resistance to fluconazole

The objective of this study was to compare phospholipase production between fluconazole-resistant and fluconazole-susceptible strains of Candida albicans in order to explore the relationship between resistance to antifungal drugs and virulence of C. albicans. Fifteen each of fluconazole-resistant (MIC ≥ 64 μg ml(-1)) and fluconazole-susceptible (MIC ≤ 8 μg ml(-1)) strains of C. albicans were incubated on egg yolk agar to detect phospholipase activity. Virulence of C. albicans was assessed by the average survival time of infected mice. Expression of phospholipase B1 mRNA and protein were detected by RT-PCR and Western blot method. Significant differences between the two groups of Candida strains were observed in phospholipase activity and average survival time of infected mice. The expression of phospholipase B1 mRNA and protein (both of secreted and intracellular forms) were higher in resistant strains than in susceptible strains. The results indicate that the phospholipase activity of C. albicans may be related to its resistance to antifungal drugs.     

The Influence of Some Antifungal Drugs on In Vitro Adherence of Candida albicans to Human Buccal Epithelial Cells

Summary:  An in vitro adherence test with 10 Candida albicans strains to buccal epithelial cells (BEC) was performed in a medium with the following antifungal drugs: nystatin, amphotericin B, 5-fluorocytosine, clotrimazole and ketoconazole. Simultaneously, an in vitro adherence test was made without drugs added, but the fungal cells had been previously treated with the same drugs. All antifungal drugs applied significantly inhibited the adherence of C. albicans to BEC (p < 0.01). Pretreatment of the fungi with drugs inhibited their adherence to BEC stronger than the addition of the drugs to the test medium. Subinhibitory doses of the drugs were less effective than therapeutic ones. The most effective inhibition of the adherence was obtained with 5-fluorocytosine and ketoconazole, while nystatin turned out to be the least effective.
Zusammenfassung:  Mit 10 Candida albicans- Stämmen wurden Adhärenzteste in vitro mit Epithelzellen der Mundschleimhaut (BEC) unter Zusatz folgender Antimyzetika durchgeführt: Nystatin, Amphotericin B, 5-Fluorcytosin, Clotrimazol und Ketoconazol. Parallel dazu wurden Adhärenzteste ohne Antimyzetika-Zusatz, jedoch nach antimyzetischer Vorbehandlung der Pilzzellen durchgeführt. Alle verwendeten Antimyzetika hemmten signifikant die Adhärenz von C. albicans an BEC (p < 0.01). Die antimyzetische Vorbehandlung hemmte die Adhärenz stärker als der Antimyzetika-Zusatz während des Adhärenzversuches. Subinhibitorische Dosen waren weniger wirksam als therapeutische Dosen. Die stärkste Adherenzhemmung wurde bei 5-FC und Ketoconazol, die schwächste bei Nystatin beobachtet.     

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.     

Effect of Amorolfine (Ro 14–4767/002) on in vitro Phagocytosis and Intracellular Killing of Cartdida albicans by Human Neutrophils: Die Wirkung von Amorolfin Po 14–4767/002) in vitro auf Phagozytose und intrazellulare Abtotung von Candida albicans durch menschlische Neutrophile

The effect of amorolfine (Ro 14-4767/002) on phagocytosis and intracellular killing of Candida albicans blastospores was determined in human neutrophil monolayer assays. At 0.2, 2 and 5 micrograms/ml amorolfine did not have any significant inhibitory or enhancing effect on phagocytosis whether following simultaneous addition of blastospores and drug to the neutrophils, prior treatment of neutrophils for 2 h before addition of blastospores or prior treatment of blastospores for 2 h. Simultaneous addition of amorolfine resulted in a significant increase in killing at all concentrations. This increase was not significantly enhanced by either preincubation of neutrophils or blastospores for 2 h with the drug.     

Addition of DNase improves the in vitro activity of antifungal drugs against Candida albicans biofilms

Cells within Candida albicans biofilms display decreased susceptibility to most clinically used antifungal agents. We recently demonstrated that extracellular DNA (eDNA) plays an important role in biofilm integrity, as a component of the biofilm matrix. This study aimed at gaining insights into the contributions of eDNA to C. albicans biofilms antifungal susceptibility by the investigation of the impact of the combined use of deoxyribonuclease I (DNase) and antifungals to treat biofilms. Candida albicans biofilms were formed using a simple and reproducible 96-well plate-based method. The activity of the combined use of 0.13 mg l(-1) DNase and antifungals was estimated using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay and total viable counts. Herein, we report the improved efficacy of amphotericin B when in combination with DNase against C. albicans biofilms, as assessed using XTT readings and viable counts. Furthermore, although DNase increased the efficacy of caspofungin in the reduction of mitochondrial activity, no changes were observed in terms of culturable cells. Deoxyribonuclease I did not affect biofilm cells susceptibility to fluconazole. This work suggests that agents that target processes affecting the biofilm structural integrity may have potential use as adjuvants of a catheter-lock therapy.     

In vitro and in vivo activity of antifungal agents in combination with fleroxacin, a new quinolone

The in vitro and in vivo interaction of fleroxacin with amphotericin B (Amph B), flucytosine (5-FC) and azoles against Candida albicans strains was tested. In vitro the interaction between fleroxacin and various antifungals was not dependent on the incubation time. Fleroxacin neither enhances nor antagonizes the in vitro activity of Amph B at high concentration (50-100 micrograms/ml). Fleroxacin has a synergistic effect with ketoconazole (KETO), but this is not observed with itraconazole (ITRA) or fluconazole (FLU). In no instance antagonism was observed. The activity of 5-FC was antagonized by fleroxacin being generally reduced by 2-4 dilution steps. In murine candidosis the efficacies of all antifungal drugs were not influenced by addition of 100 mg/kg fleroxacin. Therefore, the effects seen in in vitro tests are most probably not relevant for the clinical use of a combination of fleroxacin with antifungal drugs.     

Regulation of the Direct Fungicidal Action of Miconazole against Candida albicans ATCC 26790/Die Regulation der direkten fungiziden Wirkung von Miconazol auf Candida albicans ATCC 26790

Summary: The direct lethal action of ł 10⁻⁵ M miconazole against Candida albicans ATCC 11651 depends upon, and is therefore regulated by, growth phase, pH, and whether any miconazole is present during lag phase when susceptibility develops. Certain of these studies were recently extended to C. albicans ATCC 26790 with nearly identical results, suggesting that such regulation is a general phenomenon.
Zusammenfassung: Die direkte letale Wirkung von ≥ 10⁻⁵ M Miconazol gegen Candida albicans ATCC 11651 hängt ab und wird daher reguliert von der Wachstumsphase, dem pH-Wert und davon, ob überhaupt Miconazol während der lag-Phase anwesend ist, in der sich die Suszeptibilität entwickelt Einige dieser Untersuchungen wurden jüngst auf Candida albicans ATCC 26790 erweitert mit fast identischen Resultaten, was zu der Vorstellung führt, daß diese Regulation ein allgemeines Phänomen ist.     

Morphological changes of Candida, albicans induced by saperconazole

The effects of a new triazole antifungal agent, saperconazole, on the morphology of Candida albicans were studied by scanning and transmission electron microscopy. An inoculum of 10(6) CFU ml-1 was exposed to saperconazole at 1 and 10 micrograms ml-1 and at different times up to 24 h samples were removed for microscopic observations. The antifungal agent caused the yeasts to become round and turgescent and to cluster; budding appeared to be affected also, as seen by scanning electron microscopy. Transmission electron microscopy showed a thickened wall, the presence of intraparietal electron-dense vesicles and of multilamellae near the plasma membrane.     

The postantifungal effect and phospholipase production of oral Candida albicans from smokers,diabetics, asthmatics,denture wearers and healthy individuals following brief exposure to subtherapeutic concentrations of chlorhexidine gluconate

Candida albicans is the major aetiological agent of oral candidosis and one of its important virulent factors is the production of extracellular phospholipases, which can be modulated by subtherapeutic concentrations of antifungal agents thus decreasing their pathogenicity. Hence, considering that chlorhexidine gluconate (CG) is a common antimicrobial mouthwash used in dentistry and that its concentration in the mouth reaches subtherapeutic levels during dosage intervals due to the diluent effect of saliva and cleansing effect of the oral musculature, the postantifungal effect (PAFE) and the phospholipase production of oral C. albicans following brief exposure to subtherapeutic concentrations of CG was studied. Fifty C. albicans planktonic oral isolates obtained from smokers, diabetics, asthmatics using steroid inhalers, partial denture wearers and healthy individuals were exposed to three subtherapeutic concentrations of CG (0.005%, 0.0025% and 0.00125%) for 1 h. Isolates unexposed to CG was the control group. Thereafter the antiseptic was removed and the PAFE and phospholipase production was determined by a turbidometric method and a plate assay using an egg yolk agar medium respectively. Mean PAFE (hours) of 50 oral isolates of C. albicans following 1‐h exposure to 0.005%, 0.0025% and 0.00125% CG was 6.97, 1.85 and 0.62 respectively. The phospholipase production of these isolates was significantly suppressed with a percentage reduction of 21.68, 18.20 and 14.04% following exposure to 0.005%, 0.0025% and 0.00125% CG respectively. Brief exposure of C. albicans isolates to subtherapeutic concentrations of CG would wield an antifungal effect by suppressing growth and phospholipase production, thereby quelling its pathogenicity.     

Effectiveness of chlorhexidine on the disinfection of complete dentures colonised with fluconazole-resistant Candida albicans: in vitro study

This in vitro study evaluated different concentrations of chlorhexidine (CHX) solution on the disinfection of dentures colonised with a reference (ATCC 90028) and azole-resistant (R1, R2 e R3) strains of Candida albicans. Sterile dentures were individually inoculated with one of the strains and incubated at 37 °C for 24 h. Then, each denture was immersed in sterile saline (control) or CHX (2%, 1% or 0.2%) for 10 min. Samples of serial dilutions were spread on Agar Sabouraud Dextrose and incubated at 37 °C for 48 h. The colonies were counted and the values of log(cfu ml(-1)) were analysed by Kruskal-Wallis test (P < 0.05). Dentures immersed in CHX were incubated for 7 days. For all strains, the cfu ml(-1) values of 0.2% CHX were significantly higher than those of 2% and 1% CHX. There was no difference between the cfu ml(-1) values of 2% and 1% CHX. For dentures immersed in CHX, ATCC 90028 strain showed lower cfu ml(-1) values than R2 and R3 strains. For control dentures, cfu ml(-1) values of ATCC 90028 strain were higher than those of R strains. Immersion in 2% CHX resulted in the highest number of dentures without fungal growth after 7 days. For denture disinfection, 2% CHX was the most effective concentration, and R strains were less susceptible to disinfection. Chlorhexidine is effective in disinfection of dentures contaminated with azole-resistant C. albicans.     

In vitro investigations on the mode of action of the hydroxypyridone antimycotics rilopirox and piroctone on Candida albicans

Rilopirox and piroctone belong to the class of hydroxypyridone antimycotics. This class is not related to other antimycotics. In contrast to azole antimycotics and polyene antimycotics the mode of action of hydroxypyridone antimycotics is not fully understood. Inhibition of cellular uptake of essential compounds as well as loss of other compounds seems to be only a secondary effect of a primary not known action of these drugs. The antifungal effect in vitro depends on the medium used. The hyphal induction of Candida albicans is inhibited by hydroxypyridone antimycotics, but this effect is compensated by iron ions. A damage of the cell membrane and a direct influence on adenosine triphosphate synthesis, respectively, do not seem to be part of the mode of action. But there are clear hints that reactive oxygen species (ROS) and available metabolic activity are important parts of the mode of action of the hydroxypyridone antimycotics rilopirox and piroctone.