1887

Abstract

Summary: The development of resistance to amphotericin B methyl ester (AME), measured in terms of the amount (μg AME ml = s.r.c.) of the antibiotic required to induce a standard rate of release of K from suspensions of washed organisms, has been followed in in starved cultures under controlled conditions of aeration, pH, stirring and temperature for periods up to 120 h, and the amino acid content of the pool and the carbohydrate content of the organisms have been determined. The soluble glucan fraction of the cells decreased during the first 24 h starvation but no significant changes were observed in other carbohydrate or lipid fractions. At pH 3, the glutamate content of the pool decreased rapidly during the starvation period but other amino acids showed a general pattern of increase during the first 24 to 72 h and decrease during the later stages of starvation. Increasing the glutamate content of the pool by growth in media supplemented with sodium glutamate delayed the onset of resistance, and, in all experiments, high resistance did not develop until the glutamate content of the pool had fallen below 10 nmol (mg dry wt organisms). There was a highly significant correlation between the level of resistance and (i) the glutamate concentration in the pool and (ii) the rate of disappearance of glutamate from the pool.

Treatment of starved organisms with -ethylmaleimide (NEM) increased the resistance, the increase ranging from 10-fold for cultures starved for 24 h to 2-fold or less for cultures starved for 120 h. The resistance of NEM-treated organisms increased with the time of starvation. Treatment of organisms with β-mercaptoethanol (ESH) decreased resistance; for organisms after 24 to 96 h starvation, possessing moderate resistance (s.r.c. 15 to 20), treatment with ESH reduced this resistance to a value approximating to that of growing organisms (s.r.c. 0·1). In organisms starved for longer periods and with higher resistance, the effect of ESH was smaller and the level of ESH-insensitive resistance increased rapidly with time. Resistance thus developed in two stages; first, a stage where reduction of some components of the cell decreased resistance and, later, a stage of high resistance unaffected by reduction. Changes, similar in quality but smaller in quantity, were found in the development of resistance in organisms grown and starved at pH 7.

The activities of glutamate: NAD oxidoreductase (EC 1.4.1.2) and glutamate: NADP oxidoreductase (EC 1.4.1.3) have been estimated in extracts from organisms during growth and starvation; the NAD-linked enzyme activity remained relatively unchanged during the starvation period of 96 h while the NADP-linked enzyme activity decreased markedly during the first 24 h starvation. Estimation of the degree of reduction of the cellular NAD during the starvation period showed that AME resistance rose as the ratio of cellular NADH/NAD decreased.

It is suggested that the first stage of phenotypic resistance can be attributed to a reducible factor in the cell wall, and that the amount of this factor increases during starvation but that the developed resistance is determined by the degree of reduction of this factor. The evidence is not inconsistent with glutamate in the pool acting as a major source of reducing potential.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-109-2-191
1978-12-01
2019-10-18
Loading full text...

Full text loading...

http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-109-2-191
Loading
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error