Effects of Sublethal Concentrations of Amphotericin B on Candida albicans

This study addressed the effects of fluconazole and 5-fluorocytosine on the candidacidal activity of amphotericin B in the presence of human serum. A Candida albicans isolate that was susceptible to all three agents according to standard testing procedures was employed. Fungicidal activity was estimated by using a flow cytometric procedure that exploited the fact that yeast cells killed by amphotericin B diminish in size and take up propidium iodide. The following findings were made. (i) Fluconazole and 5-fluorocytosine each failed to inhibit pseudohyphal formation and cell aggregation even when applied at 10 and 50 ,ug/ml, respectively, for up to 10 h. Hence, these agents were not fungistatic when tested in the presence of serum. Amphotericin B, fluconazole, and 5-fluorocytosine are presently the drugs most commonly used for treating systemic infections with Candida albicans (3,13,18,22,28,32,39). Although the rnodes of action and pharmacokinetic properties of all three agents have been well studied (1, 3, 7, 21, 38, 46, 50), consensus regarding their application alone or in combination with each other is only slowly being reached. One reason for this is the difficulty in assessing the fungistatic and fungicidal activities of these agents in vitro and in vivo. Results of in vitro susceptibility tests for each agent depend heavily on many factors that must be heeded, such as composition of the medium, pH, temperature, and size of inoculum, in order for reproducible data to be obtained (15,37). Attempts to test combinations therefore encounter formidable obstacles. Furthermore, the activity of antifungal agents is almost always assessed in the absence of serum. Serum is avoided because it strongly promotes formation of pseudohyphae and cell aggregation, processes that severely impede accurate determinations of numbers of viable cells. On the other hand, systemically applied agents must perform in vivo in the presence of serum constituents and other proteins, so assessment of the activity of the agents under such conditions may not be trivial. This point has been readdressed by van Etten et al. in a recent publication (47). Amphotericin B exhibits a high degree of binding to serum components (1,(3)(4)(5), and the antifungal activity of this agent in whole serum is reduced by an order of magnitude (9,30,47). In contrast, the degree of binding of fluconazole and 5-fluorocytosine to serum components is low (7,21,46,49), and it is therefore generally assumed that results of susceptibility tests in protein-free media can be extrapolated to the situation in which serum is present. However, we are not aware of any study that has directly investigated the fungistatic effect of either fluconazole or 5-fluorocytosine on C. albicans in serum.In a previous communication, we ric assay for measuring the candidacidal activity of amphotericin B in whole human serum (25). The assay exploited the facts that C. albicans cells damaged by amphotericin B and treated with deoxycholate take up the marker dye propidium iodide, which ...

Section: Discussionsupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Antagonistic effects of fluconazole and 5-fluorocytosine on candidacidal action of amphotericin B in human serum

Martin¹,

Maier²,

Bhakdi³

1994

“…The intracellular efficacies of the antifungal agents were determined in terms of inhibition of germ tube formation as well as effective killing of C. albicans intracellularly. With AmB, inhibition of the development of germ tubes was found at concentrations lower than those required for killing of intracellular C. albicans, which has also been reported by others (25,26,38). After an increase in the ratio of infection, slightly increased AmB concentrations were required to obtain both effects.…”

Section: Discussionsupporting

confidence: 82%

Effects of amphotericin B and fluconazole on the extracellular and intracellular growth of Candida albicans

Etten

Rhee

Kampen

et al. 1991

The effects of amphotericin B and fluconazole on the extracellular and intracellular growth of Candida albicans were studied. With respect to the extracellular growth of C. albicans, antifungal activity was measured in terms of MICs and minimal fungicidal concentrations as well as by determination of the concentration that effectively killed (>99.9%) C. albicans in the absence or presence (amphotericin B only) of serum. Amphotericin B was highly active in terms of killing, even at an increased inoculum size. In the presence of serum, amphotericin B activity was substantially reduced. For fluconazole, activity was restricted to inhibition of fungal growth, even after the inoculum size was reduced. With respect to the intracellular growth of C. albicans, antifungal activity was measured by using monolayers of murine peritoneal macrophages infected with C. albicans and was measured in terms of inhibition of germ tube formation as well'as effective killing (>99%) of C. albicans. Amphotericin B was highly active against C. albicans. At an increased ratio of infection, amphotericin B activity was slightly reduced. Fluconazole had no antifungal activity. Neither a reduction in the ratio of infection nor exposure of C. albicans to fluconaz'ole prior to macrophage ingestion resulted in activity against intracellular C. albicans by fluconazole. Previous exposure of C. albicans to amphotericin B resulted in increased intracellular activity of amphotericin B. The. intracellular antifungal activity of the combination of fluconazole with amphotericin B was les than that of amphotericin B alone. Amphotericin B showed fungicidal activity against C. albicans growing both extracellularly and intracellularly, whereas fluconazole inhibited growth only of extracellular C. albicans. A slight antagonistic effect between fluconazole and aiflphotericin B was found with respect to intracellular as well as extracellular C. albicans.Deep-seated candidal infections are an important cause of morbidity and mortality in immunocompromised patients (15,21,22). The current drug of choice for most systemic mycoses is still amphotericin B (AmB), but its use is restricted by a variety of toxic side effects (6,13,15,21,22). Consequently, there is a need for effective and less toxic drugs for the treatment of patients with these infections. One of these antifungal agents is the triazole fluconazole (Flu) (4,10,14,33,34,41). In vivo experimental studies suggested that Flu is active against disseminated candidiasis (8,9,29,31,32,35,37). Recently, the antifungal efficacy of Flu was demonstrated in persistently granulocytopenic rabbits when it was used for prevention or early treatment (39,40). Little is still known, however, on the role of Flu in the treatment of systemic candidiasis in immunocompromised patients. Since the host defense mechanisms in these patients are severely impaired, the intrinsic antifungal activity of the antifungal agent is of great importance for effective treatment. Unfortunately, standardized in vitro methods for assessment of ...

“…In addition, the two lowest concentrations were below the MIC for the organism and demonstrated an impact on gene expression for each of the three genes. Previous studies with both triazoles and polyenes have demonstrated sub-MIC effects using different biologic end points (32,33,39). One explanation for the discrepancy between in vitro and in vivo investigations of the triazole postantifungal effect has been unavoidable sub-MIC exposures which are present in the latter study design (3,16).…”

Section: Discussionmentioning

confidence: 97%

Time Course of Microbiologic Outcome and Gene Expression in Candida albicans during and following In Vitro and In Vivo Exposure to Fluconazole

Lepak

Nett

Lincoln

et al. 2006

Pharmacodynamics (PD) considers the relationship between drug exposure and effect. The two factors that have been used to distinguish the PD behaviors of antimicrobials are the impact of concentration on the extent of organism killing and the duration of persistent microbiologic suppression (postantibiotic effect). The goals of these studies were (i) to examine the relationship between antimicrobial PD and gene expression and (ii) to gain insight into the mechanism of fluconazole effects persisting following exposure. Microarrays were used to estimate the transcriptional response of Candida albicans to a supra-MIC F exposure over time in vitro. Fluconazole at four times the MIC was added to a log-phase C. albicans culture, and cells were collected to determine viable growth and for microarray analyses. We identified differential expression of 18% of all genes for at least one of the time points. More genes were upregulated (n ‫؍‬ 1,053 [16%]) than downregulated (174 [3%]). Of genes with known function that were upregulated during exposure, most were related to plasma membrane/cell wall synthesis (18%), stress responses (7%), and metabolism (6%). The categories of downregulated genes during exposure included protein synthesis (15%), DNA synthesis/repair (7%), and transport (7%) genes. The majority of genes identified at the postexposure time points were from the protein (17%) and DNA (7%) synthesis categories. In subsequent studies, three genes (CDR1, CDR2, and ERG11) were examined in greater detail (more concentration and time points) following fluconazole exposure in vitro and in vivo. Expression levels from the in vitro and in vivo studies were congruent. CDR1 and CDR2 transcripts were reduced during in vitro fluconazole exposure and during supra-MIC exposure in vivo. However, in the postexposure period, the mRNA abundance of both pumps increased. ERG11 expression increased during exposure and fell in the postexposure period. The expression of the three genes responded in a dose-dependent manner. In sum, the microarray data obtained during and following fluconazole exposure identified genes both known and unknown to be affected by this drug class. The expanded in vitro and in vivo expression data set underscores the importance of considering the time course of exposure in pharmacogenomic investigations.The time course of antimicrobial activity is dependent on a drug's pharmacokinetics and two major pharmacodynamic characteristics (2, 13). The first is the rate of organism killing and whether increasing drug concentrations enhance the rate and extent of killing. The second is the presence or absence of inhibitory effects on organism growth which persist after drug levels have fallen below the MIC. These persistent effects are termed postantibiotic effects (PAE) (3,4,5,9,14,16,29,38,39,40). The PAE pharmacodynamic phenomenon underscores the importance of investigating the effects of antimicrobial exposure over time. Drugs from the triazole class demonstrate time-dependent killing but prolonged persistent effects...