Polyene Antifungal Antibiotics

ZYGMUNT, WALTER A. (Mead Johnson Research Center, Evansville, Ind.). Intracellular loss of potassium in Candida albicans after exposure to polyene antifungal antibiotics. Appl. Microbiol. 14:953-956. 1966.-Eleven antifungal antibiotics, representing three broad macrolide classes, were studied in Candida albicans for their effect on growth and on the fate of intracellular K+. Marked differences were observed among these antibiotics between their growth-inhibitory activities and their adverse effects on the integrity of the cellular membrane as evidenced by loss of K+. Antibiotics most active in inhibiting growth of C. albicans were amphotericin B, trichomycin, candidin, candicidin (all heptaenes), and nystatin (a tetraene). In addition to those antibiotics, filipin and fungichromin also caused rapid leakage of K+ from yeast cells. Interestingly, fungichromin was the least active of the 11 antibiotics in inhibiting growth. Concentrations of rimocidin 10 times as great as those required for growth inhibition caused only a slight loss in intracellular K+ after 60 min. The fungicidal properties of polyene antifungal antibiotics appear to be related to marked alterations in the permeability of the fungal cell membrane. Effects of nystatin (1, 4-8), filipin (2, 4), amphotericin B (4), and N-acetylcandidin (3) on Candida and Saccharomyces strains have been studied most extensively. Losses of such essential cellular constituents as potassium, ammonium, or inorganic phosphate ions, and of carboxylic acids, sugar phosphates, nucleotides, or protein have been shown to occur. In S. cerevisiae, loss of intraceUular K+ occurs rapidly after exposure to nystatin (7, 8) or to N-acetylcandidin (3). It seemed pertinent to extend these studies to a much larger group of polyene antibiotics and to attempt to correlate loss of intracellular K+ with the growth-inhibitory activities of these antibiotics in C. albicans. MATERIALS AND METHODS Yeast culture. C. albicans (NRRL Y477) was kindly supplied by L. J. Wickerham of the Northem Utilization Research Laboratories of the U.S. Department of Agriculture, Peoria, Ill. The culture was maintained on slants of Yeast Morphology (YM) Agar (Difco) and growo at 28 C. Growth from an 18to 24-hr agar slant was transferred into 250-ml Erlenmeyer flasks containing 50 ml of Yeast Nitrogen Base (Difco) supplemented with 1.5% dextrose (initial medium pH of 4.5) and incubated for 16 to 18 hr on a rotary shaker. Log-phase cells were obtained by inoculating fresh flasks of medium with stationary-phase culture and incubating for 3 hr on a shaker. Log-phase cells were centrifuged, washed three times in demineralized water, and adjusted to an optical density reading of 1.0 with a Coleman Junior spectrophotometer at a wavelength of 620 m1s. Dry-cell weights were also obtained on these cell suspensions. Loss of intracellular K+. In general, procedures similar to those of Harsch and Lampen (3) were used. Each 250-ml Erlenmeyer flask contained 55 mg (dry weight) of washed log-phase yeast cells in a 10-ml volume, 10 ml of 0.4 M glucose, 10 ml of 0.26 M,pH 7.5 sodium buffer (NaOH-tartaric acid-succinic acid), and 5 ml of antibiotic solution. All antibiotics were initially dissolved in 0.5 ml of dimethyl sulfoxide and diluted to an appropriate concentration with demineralized water. Demineralized water was used exclusively in the preparation of all solutions. Appropriate nonantibiotic controls contained amounts of dimethyl sulfoxide equivalent to those employed above. All flasks were incubated at 28 C on a rotary shaker and were sampled at intervals of 0, 15, 30, and 60 min. Immediately after sampling, the cell suspensions were centrifuged in chilled tubes, and the supernatant solutions were analyzed later for K+ content. A Coleman flame photometer with an oxygen-natural gas burner was used for the K+ assays. Antibiotics. The antibiotics used were kindly fur953 on N ovem er 2, 2017 by gest ht://aem .sm .rg/ D ow nladed fom