Journal of Antimicrobial Chemotherapy (1992) 29, 129-136
The postantifungal effect of 5-fluorocytosine on Candida albicans Gene M. Scalarone*, Ynzmn Mikami*, Nobnyukl Kurita*, Katsnldyo Yazawa* and Makoto Mlyaji*
The in-vitro postantifungal effect (PAFE) of 5-fluorocytosine for Candida albicans for short periods of time was investigated. Yeast cells were exposed for 0-5,1 or 2 h to a range of concentrations (0-1-3-2 mg/L) of 5-fluorocytosine. The PAFE was quantitated by determinations of the number of colony forming units at hourly intervals (0-10 h) after removal of the drug by dilution. The length of the PAFE was dependent upon the concentration of 5-fluorocytosine and the duration of exposure. An exposure time of 0-5 h resulted in PAFE's ranging from 0 to 4-2 h. Exposure times of 1 and 2 h resulted in longer PAFEs and in many instances suppression of cell growth was seen for the entire evaluation period (up to ten hours).
Introduction
During the past few years tfiere have been several studies concerned with the postantibiotic effect (PAE) of various antimicrobial agents on bacterial growth. The term PAE refers to the phenomenon of suppression of bacterial growth that persists after limited exposure to an antibiotic. Short exposures of bacteria to various antimicrobials in vitro are not followed by immediate bacterial growth after the drug has been removed, but, rather a lag phase or postantibiotic effect has been observed which can last for several hours (Gerber & Craig, 1981). It should be emphasized that the PAE is due to prior antimicrobial exposure for a short period of time rather than continued exposure for a longer time (McDonald, Craig & Kunin, 1977; Vogehnan & Craig, 1985). Investigators have generally used viable counts to determine organism regrowth following removal of the antimicrobial agent by either repeated washing, drug inactivation or by a 100- to 1000-fold dilution in fresh media (Bundtzen et al., 1981; Gerber & Craig, 1981; Vogehnan & Craig, 1985). In-vitro PAEs have been observed for Gram-positive bacteria (Cooper et al., 1990; Oshida et al., 1990; Winstanley & Hastings, 1990) as well as Gram-negative bacteria (Chadrasekar & Sluchak, 1991; Pruul & McDonald, 1990; Wu & Livermore, 1990). These represent only a few recent reports, but many other investigations on PAE of antibacterial drugs have been performed during recent years. In addition, measurements of PAE have been made in vivo after drug levels at foci of infections have fallen below the MIC (Gudmundsson, Vogehnan & Craig, 1986; Oshida et al., 1990; Vogehnan et al., 1988). Measurements of the PAE of several antimicrobials have been demonstrated with numerous bacterial organisms, however we are not aware of any such investigations 129 0305-7453/92/020129+08 $02.00/0
© 1992 The Brituh Society for Antimicrobial Chemotherapy
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'Department of Biological Sciences, Idaho State University, Pocatello, Idaho 83209, USA; bDepartment of Experimental Chemotherapy, Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chiba, Japan
130
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with fungi. Various in-vitro susceptibility testing methods for anti-fungal agents have been employed, but they have not been very reliable or readily available and have suffered from a lack of a universally accepted standardized testing procedure (Bodey, 1988). Our laboratory has therefore initiated studies to determine the effect of limited exposure of various antifungal agents on fungal cell regrowth following removal of the agent by dilution. In the present study we investigated the effect of different concentrations of 5-fluorocytosine (5-FC) on Candida albiams following exposure for varied periods of time. We have termed this phenomenon the postantifungal effect (PAFE).
Organism C. albicans strain IFM 1001 was used in this study. Yeast cells were maintained on Sabouraud's dextrose agar (Eiken Chemical Co. Ltd., Tokyo, Japan) slants and then transferred to fresh media and incubated for approximately 18 h prior to use in the assay. This strain of C. albicans has been used as the yeast cells exhibit minimal clumping activity in commonly used fungal media for colony counting, such as Sabouraud's dextrose agar and yeast nitrogen base medium. Microscopic determinations showed budding growth and suppression of colony formation was not due to excessive clumping or mycelial growth of the yeast cells. Antifungal agent The drug 5-fluorocytosine (5-FC) was provided by Nippon Roche, K.K. (Tokyo, Japan). An initial stock solution of 5-FC (1000 mg/L) was made in saline. The solution was then filter sterilized by passage through 0-22 /JM membrane filters (Nucleopore Corp., Pleasanton, CA). Concentrations of 5-FC ranging from 0-1 to 3-2 mg/L (twofold serial dilutions) were then prepared in sterile saline for use in the assay. PAFE protocol C. albicans yeast cells were harvested from the Sabouraud's dextrose agar slant and the suspension counted using a haemocytometer. The cell number was adjusted to approximately 1 x 10s cells/mL, and 0-2 mL of the suspension was added to tubes containing 1-6 mL of yeast nitrogen base medium (Difco Laboratories, Detroit, MI) plus 1% glucose. Different concentrations of 5-FC, as above, were added to each tube (0-2 mL). A control tube containing 1-6 mL of medium, 0-2 mL of yeast cells and 0-2 mL of saline was used in each experiment. Three separate experiments were performed in which each of the five 5-FC-yeast cell combinations and control suspensions were incubated at 30°C with shaking for 0-5, 1 or 2 h. The assays were performed at 30°C since studies in our laboratory and by other investigators (Pfaller et al., 1990) have indicated that this temperature was optimal for susceptibility determinations with 5-FC and C. albicans in yeast nitrogen base medium plus 1 % glucose. After incubation the antifungal agent was removed by a dilution technique in which the cultures were diluted lO'-lCP-fold in fresh medium. Counts of colony forming units (cfu) were determined on Sabouraud's dextrose agar immediately following dilution and removal of 5-FC and at hourly intervals for 8 h (2 h exposure time) or 10 h (0-5
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Materials and methods
Postantifangal effect of 5-FC on C. albicaas
131
and 1 h exposure times). Sterile saline was used for serial ten- fold dilutions as required and 50 /tL of each dilution were plated in duplicate. Plates were read after incubation for 48 h at 30°C. Calculation of PAFE
Results The PAFEs of 5-FC for C. albicans strain IFM 1001 after exposure for 0-5, 1 or 2 h were calculated from the viable count regrowth curves (Figures 1, 2 and 3) and are shown in the (Table). The duration of PAFE was prolonged by an increase in 5-FC concentration and exposure time. From the data a PAFE value was determined at the four colony number levels Gog|0 increases from 0-25 to 1-0) when the yeast cells were exposed to five concentrations of 5-FC (0-1, 0-2, 0-4, 0-8, 1-6 mg/L) for 0-5 h (Figure 1 and Table). When the exposure time was increased to 1 h, only three concentrations of
Table. PAFE of various concentrations and exposure times of 5-fluorocytosine for C. albicans Exposure timc(h)
0-1
0-25
0-5 10 20
0 0-1 ND
0 4-7 >8
06 4-8 >8
1-5 > 10 >8
2-9 > 10 >8
ND ND >8
O50
0-5 10 20
0-4 1-6 ND
0-5 > 10 >8
10 > 10 >8
1-7 > 10 >8
30 > 10 >8
ND ND >8
0-75
0-5 10 20
0-5 1-2 ND
10 >10 >8
1-5 > 10 >8
2-2 > 10 >8
4-2 >10
>8
ND ND >8
0-5 10 20
0-6 09 ND
11 > 10 >8
1-6 > 10 >8
21 >10 >8
3-9 > 10 >8
ND ND >8
Logio cfu increase
10
5-fluorocytosine concentration (mg/L) 0-2 04 08 1-6
PAFE calculated for post-exposure increase* in colony numbers of 0-25, 05, 0-75 and 1-0 log10. ND, Not done.
3-2
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A modification of the method previously described for the calculation of the postantibiotic effect with bacteria (Bundtzen et al., 1981; Gerber & Craig, 1981) was used to quantitate the effect of 5-FC on C. albicans growth and as a basis for comparison of similar studies performed on different days. The duration of PAFE was calculated from the viable count regrowth curves using the formula PAFE = T — C, where T was the time (h) required for the yeast cells in the test culture to increase by a defined number after dilution/removal of drug, and C was the equivalent time for the control culture. The PAFE was calculated for post-exposure increases of colony number of 0-25, 0-5, 075 and 1-OI^JO- Thus T—C expresses the time interval during which an antifungal agent may be shown to have a suppressive effect on fungal growth (Gerber & Craig, 1981).
132
G. M. Scmlarooe el oL
E
10 5 6 Time (h) Figure 1. Growth curves of C. aSbiewu itrain IFM 1001 following exposure to 0-1 ( • ) , 0-2 (A). 04 (A), 0-8 (D) Bad 1-6 ( • ) mg/L of 5-fluorocytosine for 05 h, and the antifungal-free control (O)-
4
5-FC (0-1, 0-2, 0-4 mg/L) gave a PAFE value at the 0-25 log10 level and a PAFE was seen with only one concentration (0-1 mg/L) at the 0-5, 0-75 and 1-0 log10 levels (Figure 2, the Table). As the concentrations of 5-FC were increased further suppression of cell growth was seen. Similar results were obtained when the cells were exposed to the various concentrations of 5-FC for 2 h (Figure 3, and Table). Regrowth was followed for 10 h (0-5 and 1 h exposure times) and 8 h (2 h exposure time). Thus, 30 min exposure to 5-FC was sufficient to produce PAFEs ranging from less than 1 h to greater than 4 h. When the cells were exposed to 5-FC for periods of 1 to 2 h, regrowth did not occur to the same level as in the experiments where the exposure time was only 30 min. Discussion 5-Fluorocytosine (5-FQ is a synthesized fluorinated pyrimidine analogue that is highly water soluble. The agent exerts its antifungal effect by interference with pyrimidine metabolism. The drug is transported into the cell and then transformed to 5-fluorouracil or to 5-fluoro-2'-deoxyuridylate (floxuridine). These metabolites inhibit the synthesis of DNA by incorporation into RNA or inhibition of thymidylate synthetase (Barriere, 1990). The agent is active against C. albicans and Cryptococcus neoformans
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 20, 2015
O O
133
Postantifangal effect of 5-FC on C. aUucani 6-0
2
3
4
5
6
7
8
9
10
Flgnc 2. Growth curves of C. aWicans (train IFM 1001 following exposure to 01 (#), 0-2 (A). 04 (A), 0-8 (D) and 1-6 ( • ) mg/L of 5-fluorocytosine for 1 h, and the antifungal-free control (O)-
and has been used for several years for the treatment of fungal infections caused by yeast-like fungi (Bodey, 1988). One of the problems, however, associated with 5-FC and other antifungal agents, is that in-vitro susceptibility testing gives variable data, primarily due to the lack of a universally accepted standardized testing methodology for fungi. In-vitro determinations of the minimum inhibitory concentration (MIC) have been performed by certain laboratories, but the number of variables associated with the procedures (e.g. media, incubation temperature, duration of incubation/exposure time, etc.) have caused great concern among clinicians and laboratory staff for several years. Mikami et al. (1987) and Mikami, Yazawa & Matsumae (1991) have performed many evaluations of the invitro antifungal activity of 5-FC and other antifungal agents on C. albicans using different media. In both studies they showed that the medium used for the assay had a profound effect on the MIC of 5-FC, with values ranging from less than 0-19 mg/L to > 100 mg/L when the assays were performed using yeast morphology agar or Sabouraud's dextrose agar, respectively. Another factor to consider regarding the determination of MIC, is that the fungi are continuously exposed to a constant level of the antifungal drug. This differs considerably from the situation in vivo where organisms are exposed to fluctuating levels of
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 20, 2015
1
134
G. M. Scalmrone et aL
FTgnrt 3. Growth curves of C. albiams strain IFM 1001 following exposure to 0-2 ( # ) , 0-4 (A). 0-8 (A). 1-6 ( • ) and 3-2 ( • ) mg/L of 5-fluorocytoiine for 2 h, and the «ntifungal-free control (O)-
drug. Thus, MIC values may be misleading with regard to the in-vivo situation during treatment. We have therefore investigated another in-vitro approach to susceptibility determination that may provide valuable information that cannot be derived from standard invitro sensitivity tests. A number of PAE studies with bacteria showed that a period of suppression of bacterial growth was induced after a short period of exposure to the antimicrobial agent. In this report we have been able to demonstrate a postantifungal effect or PAFE, when C. albicans was exposed to 5-FC. An exposure time as short as 0-5 h produced a PAFE and longer exposure times resulted in more profound suppressive effects. In addition, higher drug concentrations resulted in longer PAFEs. It is also of interest to note that the prolonged PAFEs were induced with considerably lower concentrations of 5-FC than those normally reached in serum (35-70 mg/L; Bodey, 1988) during standard dosing regimens. Therefore, this alternative in-vitro susceptibility assay provides information on the interaction between the antifungal agent and the fungal organism that standard susceptibility tests do not provide. PAFE determinations may provide the clinician with additional data required to select optimal dosing regimens. Thus, the presence or absence of a PAFE provides a theoretical rationale for either less frequent or more frequent administration of the antifungal agent. Our studies are continuing in an effort
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3-0
Postanttfnngal effect of 5-FC on C. albiaau
135
to further investigate this interesting phenomenon, using different antifungal agents and different growth evaluation parameters.
Acknowledgements
Financial support provided by the Japanese Ministry of Education for Eh- Gene M. Scalarone as a guest professor (foreign researcher) at Chiba University, Research Center for Pathogenic Fungi and Microbial Toxicoses. We are grateful for the excellent technical assistance provided by Yuri Ichihara and Shinji Ohashi.
Barriere, S. L. (1990). Pharmacology and pharmacokinetics of traditional systemic antifungal agents. Pharmacotherapy 10 (6-P»rt 3), 134S-40S. Bodcy, G. P. (1988). Topical and systemic antifungal agents. Medical Clinics of North America 72, 637-59. Bundtzen, R. W., Gerber, A. U., Conn, D. L. & Craig, W. A. (1981). Postantibiotic suppression of bacterial growth. Reviews of Infectious Diseases 3, 28-37. Chandrasekar, P. H. & Sluchak, J. A. (1991). In-vitro susceptibility of cefoperazone-susceptible and-resistant gram-negative rods to cefoperazone plus sulbactam, other beta-lactams, aminoglycosidcs and quinolone. Infection 19, 49-53. Cooper, M. A., Jin, Y. F., Ashby, J. P., Andrews, J. M. & Wise, R. (1990). In-vitro comparison of the post-antibiotic effect of vancomycin and teicoplanin. Journal of Antimicrobial Chemotherapy 26, 203-7. Gerber, A. U. & Craig, W. A. (1981). Growth kinetics of respiratory pathogens after short exposures to ampitillin and erythromycin in vitro. Journal of Antimicrobial Chemotherapy 8, Suppl. C, 81-91. Gudmundsson, S., Vogehnan, B. & Craig, W. A. (1986). The in-vivo postantibiotic effect of imipenem and other new antimicrobials. Journal of Antimicrobial Chemotherapy 18, Suppl. E. 67-73. McDonald, P. J., Craig, W. A. & Kunin, C. M. (1977). Persistent effect of antibiotics on Staphylococcus aureus after exposure for limited periods of time. Journal of Infectious Diseases 135, 217-23. Mikami, Y., Hour-Yong, C , Yazawa, K., Uno, J., Arai, T. & Kanno, H. (1987). Some new approaches to the evaluation of antifungal activities of amphotericin B. Japanese Journal of Medical Mycology 28, 373-84. Mikami, Y., Yazawa, K. & Matsumae, A. (1991). Evaluation of in vitro antifungal activities of amphotericin B, fluconazole, flucytosine, itraconazole and miconazole on seven different antifungal assay media. Chemotherapy (Tokyo) 39, 761-70. Oshida, T., Onta, T., Nakanishi, N., Matsushita, T. & Yamaguchi, T. (1990). Activity of subminimal inhibitory concentrations of aspoxicillin in prolonging the postantibiotic effect against Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 26, 29-38. Pfaller, M. A., Rinaldi, M. G., Galgjani, J. N., Bartlett, M. S., Body, B. A., Espinel-Ingroff, A. et al. (1990). Collaborative investigation of variables in susceptibility testing of yeasts. Antimicrobial Agents and Chemotherapy 34, 1648-54. Pruul, H. & McDonald, P. J. (1990). Lomefloxacin-induced modification of the kinetics of growth of gram-negative bacteria and susceptibility to phagocytic killing by human neutrophils. Journal of Antimicrobial Chemotherapy 25, 91-101. Vogehnan, B. S. & Craig, W. A. (1985). Postantibiotic effects. Journal of Antimicrobial Chemotherapy 15, Suppl. A, 37-46. Vogehnan, B. S., Gudmundsson, S., Tumidge, J., Leggett, J. & Craig, W. A. (1988). In vivo postantibiotic effect in a thigh infection in neutropenic mice. Journal of Infectious Diseases 157, 287-98. Winstanley, T. G. & Hastings, J. G. (1990). Synergy between penicillin and gentamicin against enterococci. Journal of Antimicrobial Chemotherapy 25, 551-60.
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References
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Wu, P. J. & Iivennore, D. M. (1990). Response of chemostat cultures of Pseudomonas aeruginosa to carbapenems and other /Madams. Journal of Antimicrobial Chemotherapy 25, 891-902. {Received 29 August 1991; revised version accepted 5 November 1991)
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 20, 2015
The postantifungal effect of 5-fluorocytosine on Candida albicans Gene M. Scalarone*, Ynzmn Mikami*, Nobnyukl Kurita*, Katsnldyo Yazawa* and Makoto Mlyaji*
The in-vitro postantifungal effect (PAFE) of 5-fluorocytosine for Candida albicans for short periods of time was investigated. Yeast cells were exposed for 0-5,1 or 2 h to a range of concentrations (0-1-3-2 mg/L) of 5-fluorocytosine. The PAFE was quantitated by determinations of the number of colony forming units at hourly intervals (0-10 h) after removal of the drug by dilution. The length of the PAFE was dependent upon the concentration of 5-fluorocytosine and the duration of exposure. An exposure time of 0-5 h resulted in PAFE's ranging from 0 to 4-2 h. Exposure times of 1 and 2 h resulted in longer PAFEs and in many instances suppression of cell growth was seen for the entire evaluation period (up to ten hours).
Introduction
During the past few years tfiere have been several studies concerned with the postantibiotic effect (PAE) of various antimicrobial agents on bacterial growth. The term PAE refers to the phenomenon of suppression of bacterial growth that persists after limited exposure to an antibiotic. Short exposures of bacteria to various antimicrobials in vitro are not followed by immediate bacterial growth after the drug has been removed, but, rather a lag phase or postantibiotic effect has been observed which can last for several hours (Gerber & Craig, 1981). It should be emphasized that the PAE is due to prior antimicrobial exposure for a short period of time rather than continued exposure for a longer time (McDonald, Craig & Kunin, 1977; Vogehnan & Craig, 1985). Investigators have generally used viable counts to determine organism regrowth following removal of the antimicrobial agent by either repeated washing, drug inactivation or by a 100- to 1000-fold dilution in fresh media (Bundtzen et al., 1981; Gerber & Craig, 1981; Vogehnan & Craig, 1985). In-vitro PAEs have been observed for Gram-positive bacteria (Cooper et al., 1990; Oshida et al., 1990; Winstanley & Hastings, 1990) as well as Gram-negative bacteria (Chadrasekar & Sluchak, 1991; Pruul & McDonald, 1990; Wu & Livermore, 1990). These represent only a few recent reports, but many other investigations on PAE of antibacterial drugs have been performed during recent years. In addition, measurements of PAE have been made in vivo after drug levels at foci of infections have fallen below the MIC (Gudmundsson, Vogehnan & Craig, 1986; Oshida et al., 1990; Vogehnan et al., 1988). Measurements of the PAE of several antimicrobials have been demonstrated with numerous bacterial organisms, however we are not aware of any such investigations 129 0305-7453/92/020129+08 $02.00/0
© 1992 The Brituh Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 20, 2015
'Department of Biological Sciences, Idaho State University, Pocatello, Idaho 83209, USA; bDepartment of Experimental Chemotherapy, Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chiba, Japan
130
G. M. Scalarone et aL
with fungi. Various in-vitro susceptibility testing methods for anti-fungal agents have been employed, but they have not been very reliable or readily available and have suffered from a lack of a universally accepted standardized testing procedure (Bodey, 1988). Our laboratory has therefore initiated studies to determine the effect of limited exposure of various antifungal agents on fungal cell regrowth following removal of the agent by dilution. In the present study we investigated the effect of different concentrations of 5-fluorocytosine (5-FC) on Candida albiams following exposure for varied periods of time. We have termed this phenomenon the postantifungal effect (PAFE).
Organism C. albicans strain IFM 1001 was used in this study. Yeast cells were maintained on Sabouraud's dextrose agar (Eiken Chemical Co. Ltd., Tokyo, Japan) slants and then transferred to fresh media and incubated for approximately 18 h prior to use in the assay. This strain of C. albicans has been used as the yeast cells exhibit minimal clumping activity in commonly used fungal media for colony counting, such as Sabouraud's dextrose agar and yeast nitrogen base medium. Microscopic determinations showed budding growth and suppression of colony formation was not due to excessive clumping or mycelial growth of the yeast cells. Antifungal agent The drug 5-fluorocytosine (5-FC) was provided by Nippon Roche, K.K. (Tokyo, Japan). An initial stock solution of 5-FC (1000 mg/L) was made in saline. The solution was then filter sterilized by passage through 0-22 /JM membrane filters (Nucleopore Corp., Pleasanton, CA). Concentrations of 5-FC ranging from 0-1 to 3-2 mg/L (twofold serial dilutions) were then prepared in sterile saline for use in the assay. PAFE protocol C. albicans yeast cells were harvested from the Sabouraud's dextrose agar slant and the suspension counted using a haemocytometer. The cell number was adjusted to approximately 1 x 10s cells/mL, and 0-2 mL of the suspension was added to tubes containing 1-6 mL of yeast nitrogen base medium (Difco Laboratories, Detroit, MI) plus 1% glucose. Different concentrations of 5-FC, as above, were added to each tube (0-2 mL). A control tube containing 1-6 mL of medium, 0-2 mL of yeast cells and 0-2 mL of saline was used in each experiment. Three separate experiments were performed in which each of the five 5-FC-yeast cell combinations and control suspensions were incubated at 30°C with shaking for 0-5, 1 or 2 h. The assays were performed at 30°C since studies in our laboratory and by other investigators (Pfaller et al., 1990) have indicated that this temperature was optimal for susceptibility determinations with 5-FC and C. albicans in yeast nitrogen base medium plus 1 % glucose. After incubation the antifungal agent was removed by a dilution technique in which the cultures were diluted lO'-lCP-fold in fresh medium. Counts of colony forming units (cfu) were determined on Sabouraud's dextrose agar immediately following dilution and removal of 5-FC and at hourly intervals for 8 h (2 h exposure time) or 10 h (0-5
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Materials and methods
Postantifangal effect of 5-FC on C. albicaas
131
and 1 h exposure times). Sterile saline was used for serial ten- fold dilutions as required and 50 /tL of each dilution were plated in duplicate. Plates were read after incubation for 48 h at 30°C. Calculation of PAFE
Results The PAFEs of 5-FC for C. albicans strain IFM 1001 after exposure for 0-5, 1 or 2 h were calculated from the viable count regrowth curves (Figures 1, 2 and 3) and are shown in the (Table). The duration of PAFE was prolonged by an increase in 5-FC concentration and exposure time. From the data a PAFE value was determined at the four colony number levels Gog|0 increases from 0-25 to 1-0) when the yeast cells were exposed to five concentrations of 5-FC (0-1, 0-2, 0-4, 0-8, 1-6 mg/L) for 0-5 h (Figure 1 and Table). When the exposure time was increased to 1 h, only three concentrations of
Table. PAFE of various concentrations and exposure times of 5-fluorocytosine for C. albicans Exposure timc(h)
0-1
0-25
0-5 10 20
0 0-1 ND
0 4-7 >8
06 4-8 >8
1-5 > 10 >8
2-9 > 10 >8
ND ND >8
O50
0-5 10 20
0-4 1-6 ND
0-5 > 10 >8
10 > 10 >8
1-7 > 10 >8
30 > 10 >8
ND ND >8
0-75
0-5 10 20
0-5 1-2 ND
10 >10 >8
1-5 > 10 >8
2-2 > 10 >8
4-2 >10
>8
ND ND >8
0-5 10 20
0-6 09 ND
11 > 10 >8
1-6 > 10 >8
21 >10 >8
3-9 > 10 >8
ND ND >8
Logio cfu increase
10
5-fluorocytosine concentration (mg/L) 0-2 04 08 1-6
PAFE calculated for post-exposure increase* in colony numbers of 0-25, 05, 0-75 and 1-0 log10. ND, Not done.
3-2
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 20, 2015
A modification of the method previously described for the calculation of the postantibiotic effect with bacteria (Bundtzen et al., 1981; Gerber & Craig, 1981) was used to quantitate the effect of 5-FC on C. albicans growth and as a basis for comparison of similar studies performed on different days. The duration of PAFE was calculated from the viable count regrowth curves using the formula PAFE = T — C, where T was the time (h) required for the yeast cells in the test culture to increase by a defined number after dilution/removal of drug, and C was the equivalent time for the control culture. The PAFE was calculated for post-exposure increases of colony number of 0-25, 0-5, 075 and 1-OI^JO- Thus T—C expresses the time interval during which an antifungal agent may be shown to have a suppressive effect on fungal growth (Gerber & Craig, 1981).
132
G. M. Scmlarooe el oL
E
10 5 6 Time (h) Figure 1. Growth curves of C. aSbiewu itrain IFM 1001 following exposure to 0-1 ( • ) , 0-2 (A). 04 (A), 0-8 (D) Bad 1-6 ( • ) mg/L of 5-fluorocytosine for 05 h, and the antifungal-free control (O)-
4
5-FC (0-1, 0-2, 0-4 mg/L) gave a PAFE value at the 0-25 log10 level and a PAFE was seen with only one concentration (0-1 mg/L) at the 0-5, 0-75 and 1-0 log10 levels (Figure 2, the Table). As the concentrations of 5-FC were increased further suppression of cell growth was seen. Similar results were obtained when the cells were exposed to the various concentrations of 5-FC for 2 h (Figure 3, and Table). Regrowth was followed for 10 h (0-5 and 1 h exposure times) and 8 h (2 h exposure time). Thus, 30 min exposure to 5-FC was sufficient to produce PAFEs ranging from less than 1 h to greater than 4 h. When the cells were exposed to 5-FC for periods of 1 to 2 h, regrowth did not occur to the same level as in the experiments where the exposure time was only 30 min. Discussion 5-Fluorocytosine (5-FQ is a synthesized fluorinated pyrimidine analogue that is highly water soluble. The agent exerts its antifungal effect by interference with pyrimidine metabolism. The drug is transported into the cell and then transformed to 5-fluorouracil or to 5-fluoro-2'-deoxyuridylate (floxuridine). These metabolites inhibit the synthesis of DNA by incorporation into RNA or inhibition of thymidylate synthetase (Barriere, 1990). The agent is active against C. albicans and Cryptococcus neoformans
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 20, 2015
O O
133
Postantifangal effect of 5-FC on C. aUucani 6-0
2
3
4
5
6
7
8
9
10
Flgnc 2. Growth curves of C. aWicans (train IFM 1001 following exposure to 01 (#), 0-2 (A). 04 (A), 0-8 (D) and 1-6 ( • ) mg/L of 5-fluorocytosine for 1 h, and the antifungal-free control (O)-
and has been used for several years for the treatment of fungal infections caused by yeast-like fungi (Bodey, 1988). One of the problems, however, associated with 5-FC and other antifungal agents, is that in-vitro susceptibility testing gives variable data, primarily due to the lack of a universally accepted standardized testing methodology for fungi. In-vitro determinations of the minimum inhibitory concentration (MIC) have been performed by certain laboratories, but the number of variables associated with the procedures (e.g. media, incubation temperature, duration of incubation/exposure time, etc.) have caused great concern among clinicians and laboratory staff for several years. Mikami et al. (1987) and Mikami, Yazawa & Matsumae (1991) have performed many evaluations of the invitro antifungal activity of 5-FC and other antifungal agents on C. albicans using different media. In both studies they showed that the medium used for the assay had a profound effect on the MIC of 5-FC, with values ranging from less than 0-19 mg/L to > 100 mg/L when the assays were performed using yeast morphology agar or Sabouraud's dextrose agar, respectively. Another factor to consider regarding the determination of MIC, is that the fungi are continuously exposed to a constant level of the antifungal drug. This differs considerably from the situation in vivo where organisms are exposed to fluctuating levels of
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 20, 2015
1
134
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FTgnrt 3. Growth curves of C. albiams strain IFM 1001 following exposure to 0-2 ( # ) , 0-4 (A). 0-8 (A). 1-6 ( • ) and 3-2 ( • ) mg/L of 5-fluorocytoiine for 2 h, and the «ntifungal-free control (O)-
drug. Thus, MIC values may be misleading with regard to the in-vivo situation during treatment. We have therefore investigated another in-vitro approach to susceptibility determination that may provide valuable information that cannot be derived from standard invitro sensitivity tests. A number of PAE studies with bacteria showed that a period of suppression of bacterial growth was induced after a short period of exposure to the antimicrobial agent. In this report we have been able to demonstrate a postantifungal effect or PAFE, when C. albicans was exposed to 5-FC. An exposure time as short as 0-5 h produced a PAFE and longer exposure times resulted in more profound suppressive effects. In addition, higher drug concentrations resulted in longer PAFEs. It is also of interest to note that the prolonged PAFEs were induced with considerably lower concentrations of 5-FC than those normally reached in serum (35-70 mg/L; Bodey, 1988) during standard dosing regimens. Therefore, this alternative in-vitro susceptibility assay provides information on the interaction between the antifungal agent and the fungal organism that standard susceptibility tests do not provide. PAFE determinations may provide the clinician with additional data required to select optimal dosing regimens. Thus, the presence or absence of a PAFE provides a theoretical rationale for either less frequent or more frequent administration of the antifungal agent. Our studies are continuing in an effort
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Postanttfnngal effect of 5-FC on C. albiaau
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to further investigate this interesting phenomenon, using different antifungal agents and different growth evaluation parameters.
Acknowledgements
Financial support provided by the Japanese Ministry of Education for Eh- Gene M. Scalarone as a guest professor (foreign researcher) at Chiba University, Research Center for Pathogenic Fungi and Microbial Toxicoses. We are grateful for the excellent technical assistance provided by Yuri Ichihara and Shinji Ohashi.
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