We have tompared the activities of fluconazole, and ketoconazole against ketoconazole-susceptible and -resistant strains of Candida albicans in a neutropenic-site rabbit model. Oral treatment with fluconazole resulted in much higher serum and extravascular concentrations of this antifungal agent than did comparable doses of ketoconazole. Fluconazole had no additional in vivo activity against the ketoconazole-susceptible strains; no fungicidal activity was observed with peak drug levels as high as -75 ,ug/ml in the infection sites. Significant fungistatic activity against the ketoconazole-resistant strains was observed with fluconazole treatment (80 mg/kg), but not with less fluconazole (20 mg/kg) or with ketoconazole (-67 mg/kg). In vitro susceptibility tests separated the ketoconazole-susceptible strains from the ketoconazole-resistant strains, but the results were variable when the resistant strains were tested with fluconazole.Fluconazole (UK 49,858) is a bis-triazole antifungal agent that is active when administered both orally and parenterally. Pharmacologically, the drug is characterized by excellent oral absorption, high serum and extravascular levels, and a long half-life that allows once-daily administration (8). These properties may explain why fluconazole has appeared to be more active than ketoconazole in some studies of experimental infections with Candida albicans despite its apparently lower in vitro activity (16; K. Richardson, R. J. Andrews, T. T. Howarth, M. S. Marriott, M. H. Tarbit, and P. F. Troke, Program Abstr. 25th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 813, 1985). However, previous animal studies (10,12,15) have not shown any activity of fluconazole against strains of C. albicans which have become resistant to ketoconazole, i.e., strains obtained from patients with chronic mucocutaneous candidiasis who relapsed during prolonged oral therapy with ketoconazole (4, 13).In the present study we investigated the activities of fluconazole and ketoconazole against ketoconazole-susceptible and -resistant C. albicans strains, using semipermeable Visking chambers (molecular weight cutoff, 15,000) implanted subcutaneously in rabbits as the infection sites. The study was designed to use doses of fluconazole that yield much higher fluconazole levels than those attainable with ketoconazole to determine whether fluconazole is fungicidal or active against the ketoconazole-resistant strains. In vitro susceptibility tests were also performed to further assess the correlation with the in vivo studies. with shaking at 150 rpm, the cultures were then diluted in broth media to concentrations of _105 CFU/ml as determined by plate counts, and 1-ml aliquots were added to the drug dilutions. For each yeast strain, the dilutions were set up in triplicate; in addition, the tests were repeated one or more times on different dates. After 24 and 48 h of incubation at 37°C, the tubes were read turbidimetrically with a Coleman 295 spectrophotometer at 540 nm, using drug-free medium as a blank. The 24-a...

Amphotericin B and rifampin act synergistically against certain yeasts in vitro. Whether this synergism is a general phenomenon or whether the effect has strict species and strain requirements was studied. Included in a survey of the genus Candida were eight human isolates of Candida albicans and one strain each of Candida krusei, Candida tropicalis, Candida pseudotropicalis, Candida parapsilosis, Candida guilliermodnii, and Candida stellatoidea. Cultures in both control and drug-containing liquid medium were incubated at 37 C with aeration. Effects of the drugs were determined from viability assays performed at zero-time and at 17 hr. Amphotericin C and rifampin were judged to be synergistic if any one of three different sets of criteria was met. Combined activity greater than the sums of individual drug effects was required in each set of criteria. Partially inhibitory or fungistatic levels of amphotericin B and noninhibitory concentrations of rifampin acted synergistically against all strains of Candida examined. Within the genus Candida, synergism of amphotericin B and rifampin appears to be a rather general phenomenon.

A direct relationship between the concentration-dependent rate of amphotericin B-induced K+ release from Candida albicans and the concentration-dependent rate of killing by the drug was established. This relationship together with the observed rapidity of both release and killing action supports the conclusion that the lethal action of amphotericin B is primarily physicochemical in nature.There is abundant evidence to suggest that if amphotericin B is to kill susceptible fungal organisms, it must bind, via hydrophobic interaction, to ergosterol in the cell membrane (2,5,7, 10). This interaction presumably causes distortion and structural rearrangement to an extent that the membrane is destabilized and cannot function normally. Abnormal function is evidenced, for example, by efflux of cellular potassium ions (K+). The action is basically physicochemical in nature, and although it is generally considered to be the primary mode of amphotericin B action, it might not be the only mechanism involved or even the primary mechanism for that matter. Brajtburg and coworkers (2) have raised the possibility that the lethal action of amphotericin B involves something more than simply disturbance in membrane permeability. They found (2), as have others (1, 3), that K+ release per se is not a lethal event. In essence, their hypothesis is that subsequent to hydrophobic binding to ergosterol, amphotericin B undergoes autoxidation, with the production of reactive intermediates (e.g., free radicals and peroxides) that cause lethal oxidative damage to fungal cells. Since autoxidation of amphotericin B requires some significant period of time (8), inherent in this proposal is a series of time-dependent chemical changes preceding the lethal cell-damaging interaction. In this study the direct physicochemical cell membrane damage hypothesis has been examined further.Candida albicans 11651 was purchased from the American Type Culture Collection, Rockville, Md., and maintained on slants of Sabouraud dextrose agar (Difco Laboratories, Detroit, Mich.). Prior to each experiment, yeast cells were grown at 35 to 37°C with rotary shaking (150 rpm) in 20-ml volumes of a filter-sterilized (pH 7) synthetic broth containing 6.7 g of yeast nitrogen base (Difco), 1.5 g of L-asparagine, and 10 g of glucose in 1 liter of deionized water. Amphotericin B was purchased from Sigma Chemical Co., St. Louis, Mo., and working solutions were prepared in Me2SO at 100 times the final concentrations required for particular determinations.To prepare cells for an experiment, 4-ml volumes of a culture grown to early stationary phase (18 h) were diluted into flasks containing 16 ml of fresh medium and were incubated for 2 h with shaking at 35 to 37°C. Cells were collected by filtration, washed three times with several milliliters of saline, and suspended in 0.1 M NaCl (as recommended by the K+ electrode manufacturer, Orion Research) at -3 x 107 CFU/ ml. Forty milliliters of the suspension was then placed in either a 125-ml Erlenmeyer flask or a 100-ml beaker.In each o...

The antifungal imidazoles miconazole and ketoconazole inhibit synthesis of essential cell membrane components. Furthermore, miconazole can exert direct physicochemical cell membrane damage at relatively high levels, but ketoconazole cannot. Experiments were designed to explain our previous observation that concentrations of miconazole capable of causing direct membrane damage were no more active against Candida albicans than equimolar levels of ketoconazole. When stationary-phase cells were inoculated into medium containing either drug at 3.8 x i0-M, fungistatic effects were indistinguishable. If, however, such cultures were incubated 3 h before drug addition, differences were remarkable. After 3 h, miconazole caused a 99% reduction in CFU per milliliter within 20 min, but ketoconazole again was only fungistatic. The immediate onset, rapidity, and magnitude of the miconazole effect were indicative of direct lethal cell damage. Miconazole concentrations as low as 1.0 x 10-5 M were similarly active. It was concluded that C. albicans undergoes phenotypic changes during the growth cycle that coincidentally confer susceptibility or resistance to the lethal direct membrane damage effect of miconazole. The fungistatic or metabolic effects of ketoconazole or low-level miconazole appeared to be independent of growth phase.Antifungal imidazole-containing drugs can inhibit biosynthesis of essential fungal cell membrane components (10,12,(15)(16)(17) and exert direct physicochemical cell membrane damage unrelated to any metabolic event (5,8, 11,14,18). Each effect has been offered as the primary mechanism by which imidazoles disrupt membrane permeability. A possible reconciliation of these hypotheses is seen in some recent studies with Saccharomyces cerevisiae (12, 13). Low levels of miconazole (MCZ) inhibited synthesis of membrane components, and this was correlated with fungistatic activity. At relatively high concentrations the drug exerted direct membrane damage and was fungicidal. However, in parallel studies with ketoconazole (KCZ), the most promising of the newer imidazoles, a striking difference was noted (13). Although KCZ inhibited synthesis of membrane components and was fungistatic, it failed to show a capacity for direct membrane damage. Thus, at relatively high concentrations, MCZ exerted much greater antifungal activity than KCZ. From recent experiments with strains of Candida albicans and Candida parapsilosis, we obtained results supporting the existence of a basic difference between the drugs with respect to direct membrane damage potential (2, 3). However, we were unable to show that MCZ is more active than an equimolar concentration of KCZ against growing cultures (1, 3). In view of studies showing that in stationary phase C. albicans develops phenotypic resistance to the lethal action of MCZ (5, 9), the physiological state of our inoculum cells might explain the apparent inability of MCZ to cause direct membrane damage and death of C. albicans in a growth environment (1). This question was examined. MATE...

Studies were designed to determine whether ionic strength (mu) is a significant factor in salt inhibition of aminoglycoside action against Escherichia coli and Pseudomonas aeruginosa. In both nutrient broth (a low mu medium) and Mueller-Hinton broth (a relatively high mu medium), protection of E. coli from dihydrostreptomycin or gentamicin action by MgCl2, NaCl, or Na2SO4 was attributed to ionic strength alone. The percentage of protection increased with ionic strength and was independent of the particular salt used. Antagonism of aminoglycoside action against P. aeruginosa appeared to involve both a specific, divalent caption-dependent mechanism, revealed in Mueller-Hinton broth, and a nonspecific, ionic strength effect, elicited by sodium salts in nutrient broth. With media of relatively low salt content, variation in ionic strength itself over a range of mu of 0.02-0.14 significantly influences the effectiveness of aminoglycoside antibiotics against E. coli and P. aeruginosa.