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Antimicrobial Agents and Chemotherapy, May 2001, p. 1589-1590, Vol. 45, No. 5
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.5.1589-1590.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Effect of Potassium on Saccharomyces
cerevisiae Resistance to Fluconazole
Carlos A.
Stella* and
Hilda I.
Burgos
Departamento de Bioquímica Humana,
Facultad de Medicina, Universidad de Buenos Aires, 1121 Buenos
Aires, Argentina
Received 13 December 2000/Returned for modification 4 January
2001/Accepted 15 February 2001
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ABSTRACT |
Susceptibility of strain S288c of Saccharomyces
cerevisiae to fluconazole was assayed in the presence and absence
of KCl. Addition of 150 mM KCl renders the strain more sensitive to the antifungal agent. The effect is caused by the K+ ion rather
than the anion or the osmolarity of the medium. The increase in
sensitivity does not modify the values of intracellular and
extracellular pH established in the presence of KCl.
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TEXT |
Fluconazole is a widely used
antifungal agent, especially in the treatment of invasive mycosis. The
generation of resistant mutants operates against the effectiveness of
fluconazole treatment (1, 5, 7). Consequently, it is
important to study the conditions under which resistance to the
antifungal emerges impaired or diminished.
The potassium ion is normally found in Saccharomyces
cerevisiae growth media. However, in concentrations of 150 mM or
higher, it depolarizes the cellular membrane of this organism (3,
4). Bearing this fact in mind, we investigated the effect of
potassium ions on the susceptibility of S. cerevisiae strain
S288c to fluconazole.
Number of colonies.
S. cerevisiae strain S288c was
grown to 1.5 × 108 cells/ml on SD synthetic medium
(Difco Laboratories, Detroit, Mich.) containing 2% (wt/vol)
D-glucose. To study the effect of KCl using solid SD, an
aliquot from a preculture grown for 24 h at 30°C was diluted and
used to inoculate plates to obtain 300 to 500 colonies per plate.
Plates were incubated at 30°C, and at 72 h the number of colonies was
recorded. With 10 µg of fluconazole per ml and 150 mM KCl,
fluconazole had only a slight effect on the number of colonies.
However, the inhibition increased at a concentration of 20 µg/ml
(Fig. 1).
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FIG. 1.
Fluconazole inhibition of strain S288c on solid SD
medium containing 150 mM KCl. Values are from one representative
experiment.
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Halo formation.
To determine whether the difference observed
with KCl is due to an osmotic effect or to the presence of chloride
ions, parallel assays were carried out using sorbitol and
MgCl2. From a suspension of 1.0 × 106
cells/ml, 200-µl portions were dispersed onto plates of SD medium supplemented with 300 mM sorbitol, 150 mM KCl, or 100 mM
MgCl2. Glass-fiber filters (Schleicher & Schuell, Inc.,
catalog no. 3362), 8 mm in diameter, were impregnated with 25-µg
portions of fluconazole and placed in the center of the seeded plates.
After incubation at 30°C for 48 h, the diameters of the
inhibition halos were measured and compared (Fig.
2). The largest halo was formed on the
plate containing KCl. In the medium with MgCl2 the halo
diameter was similar to that of the control. However, in this medium
roughly 200 to 250 small colonies appeared inside the halo; they are
not visible in the photograph. Similar results were obtained using lower KCl concentrations, ranging from 50 to 100 mM.
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FIG. 2.
(Top) From left to right, halos from control and 300 mM
sorbitol-, 150 mM KCl-, and 100 mM MgCl2-supplemented
media. Photographs were taken after 48 h of incubation at 30°C.
Filters contained 25 µg of fluconazole. (Bottom) Sensitivity reported
as halo radius.
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Internal and external pHs.
The potassium ion is able to
produce intracellular alkalinization concomitantly with extracellular
acidification (2, 6). A possible explanation for the
increase in susceptibility observed with KCl is that fluconazole may
interfere with processes that release protons from the intracellular space.
To exclude this possibility, cells were incubated with 150 mM KCl, with
and without the addition of fluconazole, and intracellular
pH was
assayed with fluorescein diacetate (FDA) (
8). By assuming
a medium volume of 1.0 ml below the halo (Fig.
2), the fluconazole
concentration may be estimated to be 25 µg/ml. This concentration
was
used for the FDA assay. A suspension of 3.0 mg (dry weight)
of yeast
per ml in 4 mM potassium phthalate buffer (pH 4.5) was
incubated with
150 mM KCl and 0.13 mM FDA with and without 25
µg of fluconazole per
ml for 30 min at 30°C. Following incubation,
the mixture was
centrifuged and the precipitate was suspended
in 1.0 ml of the buffer.
After measuring the fluorescence using
50 µl of this suspension, the
pH value was interpolated using
a calibration curve. The curve was
determined by adding an aliquot
from hydrolyzed FDA to 1.8-ml portions
of solutions with different
pH values. The extracellular pH of the
supernatant of a suspension
slightly buffered with 4 mM potassium
phthalate (pH = 4.5) was
determined by using a pH
electrode.
For this purpose, 13.3 mg (dry weight) of yeast/ml was incubated with
150 mM KCl, with and without the addition of 100 µg
of fluconazole
per ml. The fluconazole concentration was increased
to maintain the
same ratio of fluconazole/mg of yeast as that
used for internal pH
measurement. After centrifugation, the pH
value of the supernatant
fluid was determined. The pH values obtained
did not show differences,
regardless of whether the cells were
incubated with or without
fluconazole (Table
1). Therefore, it
can
be excluded that fluconazole alters the alkalinization and
acidification processes elicited by the ion.
The results show that the potassium ion increases the susceptibility of
S. cerevisiae strain S288c to fluconazole. The elucidation
of the mechanism by which the ion exercises this effect needs
to be
established through future
investigations.
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FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Bioquímica Humana, Facultad de Medicina, Universidad de Buenos
Aires, Paraguay 2155 Piso 5, 1121 Buenos Aires, Argentina. Fax:
4508-3672, ext. 31. E-mail: cstella{at}fmed.uba.ar.
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Antimicrobial Agents and Chemotherapy, May 2001, p. 1589-1590, Vol. 45, No. 5
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.5.1589-1590.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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