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Reduced susceptibility in laboratory-selected mutants of Aspergillus fumigatus to itraconazole due to decreased intracellular accumulation of the antifungal agent
International Journal of Antimicrobial Agents 12 (1999) 213 – 219 www.elsevier.com/locate/isc
Original article
Reduced susceptibility in laboratory-selected mutants of Aspergillus fumigatus to itraconazole due to decreased intracellular accumulation of the antifungal agent Elias K. Manavathu *, Jose A. Vazquez, Pranatharthi H. Chandrasekar Di6ision of Infectious Diseases, Department of Medicine, Wayne State Uni6ersity School of Medicine, Wayne State Uni6ersity, Detroit, MI 48201, USA Received 16 July 1998; accepted 6 November 1998
Abstract To study the mechanism of resistance of Aspergillus fumigatus to itraconazole, spontaneous mutants with reduced susceptibility were selected by spreading 2 ×108 conidia from a clinical isolate (W73355) susceptible to miconazole (MIC 2 mg/l), itraconazole (MIC 0.25 mg/l) and amphotericin B (MIC 0.5 mg/l) on 40 peptone yeast extract glucose agar plates containing miconazole (32 mg/l). The 19 colonies that grew (frequency 0.95 ×10 − 7) in the presence of miconazole were screened by broth macrodilution technique for their susceptibility to itraconazole. A total of two isolates (frequency 1 × 10 − 8) MCZ14 and MCZ15 had MICs of 16 and 8 mg/l, respectively, for itraconazole. Both MCZ14 and MCZ15 showed concomitant increases in MICs for ketoconazole and miconazole, but not for amphotericin B. Growth inhibition studies as well as kill curve experiments revealed that MCZ14 and MCZ15 were less susceptible to itraconazole compared to the parental strain. The intracellular accumulation of itraconazole in A. fumigatus was time and concentration dependent. Maximum accumulation was obtained within 30 min at 5 mM itraconazole. In MCZ14 and MCZ15 intracellular accumulation of [3H]itraconazole was reduced by approximately 80 and 60%, respectively, compared to the susceptible parent. The respiratory inhibitor carbonyl cyanide m-chlorophenyl hydrazone at 200 mM reduced the intracellular accumulation of itraconazole by approximately 36.2% (P 50.05) in the parent and in the mutant strains. These results suggest that (i) the reduced accumulation of itraconazole in MCZ14 and MCZ15 is due to diminished permeability of the drug and perhaps not due to efflux, (ii) the uptake of itraconazole in A. fumigatus may be an energy dependent process, and (iii) decreased accumulation of itraconazole is at least in part responsible for the reduced susceptibility of the mutant isolates to itraconazole. © 1999 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Itraconazole; Aspergillus fumigatus; Drug resistance; Antifungal agents; Resistant mutants
1. Introduction Among various azoles approved as antifungal agents for clinical use, miconazole and itraconazole are the most effective agents against Aspergillus fumigatus. In vitro susceptibility studies have shown that itraconazole is approximately eight times more active against A. * Corresponding author. Present address: Department of Internal Medicine, 427 Lande Building, Wayne State University, 550 E. Canfield, Detroit, MI 48201, USA. Tel.: +1-313-5771931; fax: + 1313-9930302. E-mail address: [email protected] (E.K. Manavathu)
fumigatus than miconazole [1]. Although emergence of resistance to these antifungal agents has been found in pathogenic yeasts such as Candida albicans [2–7], few reports [8–10] have been published in the literature on azole resistance in A. fumigatus. Thus, the mechanism of resistance to azoles in filamentous fungi and in particular in A. fumigatus is not known. We recently screened approximately 200 clinical isolates of A. fumigatus [11] in order to obtain isolates that demonstrated reduced susceptibility to itraconazole. No A. fumigatus clinical isolate with an MIC greater than 4 mg/l for itraconazole was obtained. We therefore sought to iso-
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late A. fumigatus resistant to itraconazole in the laboratory. In this paper we describe the selection, in vitro susceptibility and studies on the accumulation of itraconazole in two mutant isolates of A. fumigatus that showed reduced susceptibility to itraconazole.
2. Materials and methods
2.1. Selection of itraconazole-resistant mutants A. fumigatus W73355, a clinical isolate obtained from the Microbiology Laboratory of the Detroit Medical Center, served as the parental strain in this investigation. A working culture of this strain was maintained on peptone yeast extract glucose (PYG: peptone 1 g; yeast extract 1 g; glucose 3 g; per liter of distilled water) agar slants at room temperature. For the preparation of conidial suspension, a culture of A. fumigatus W73355 was grown on PYG agar for 6 days at 35°C, and the conidia were collected as described previously [12]. Approximately 5×106 conidia per plate were spread on 40 PYG agar plates containing 32 mg miconazole/l. The plates were incubated for 6 days at 35°C in plastic sleeves. A total of 19 colonies that grew on PYG agar in the presence of miconazole were subcultured as individual isolates and subsequently screened on PYG agar containing itraconazole (16 mg/l) for their susceptibility. Of the 19 miconazole selected isolates, only MCZ14 and MCZ15 grew in the presence of itraconazole on PYG agar and were used for subsequent studies.
2.2. MIC determination The in vitro susceptibility of the parent and the mutant isolates of A. fumigatus to various antifungals was examined by determining the MIC of each compound using a previously described broth macrodilution technique [12,13] in both RPMI 1640 and PYG broth. Briefly, fresh conidia were resuspended in growth medium at a density of 2×104 conidia per ml. Then two times the required concentrations of the drugs were prepared in PYG or RPMI 1640 medium (0.5 ml) by serial dilution in sterile 6-ml polystyrene tubes (Falcon 2054) and inoculated with an equal volume (0.5 ml) of the conidial suspension prepared in the same medium. The tubes were incubated at 35°C for 48 h and scored for visible growth after vortexing the tubes gently, or scraping the walls of the tube followed by vortexing. The MIC was defined as the lowest concentration of the drug in which no visible growth occurred. The MIC determination for each isolate was repeated three times, and the data were within 91 dilution.
2.3. Growth inhibition study Cultures of W73355, MCZ14 and MCZ15 were grown in 100 ml PYG medium from conidia (1 ×104/ ml) in the presence of 0–4 mg/l itraconazole at 35°C for 48 h with gentle agitation (90 rpm) on an orbital shaker. The mycelia were collected on Whatman no. 2 filter paper and washed once with 250 ml sterile distilled water. The washed mycelia were harvested by vacuum-filtration and the wet weight was determined. The wet weight of the mycelia was plotted against the concentrations of itraconazole used for the susceptible and the resistant isolates to examine growth inhibition.
2.4. Kill cur6es In the presence of various concentrations of itraconazole (0–4 mg/l), 5-ml aliquots of a conidial suspension prepared in PYG broth (1× 106 conidia/ml) were incubated at 25°C. At various time intervals 0.1-ml aliquots of the conidial suspension were removed, diluted 102 – 104 fold, and 0.1-ml quantities were spread in duplicate on PYG agar plates. The plates were incubated at 35°C for 48 h and the number of colony forming units (c.f.u.) per ml of conidial suspension were calculated and plotted against the time of exposure to itraconazole for the construction of the kill curve.
2.5. Itraconazole uptake studies A. fumigatus cultures were grown from conidia (1× 104 conidia/ml) in 100 ml of PYG broth at 35°C for 48 h on a gyratory shaker (170 rpm). The mycelia were harvested by filtration using a Buchner funnel on Whatman no. 2 filter paper, washed with sterile distilled water (500 ml) and gently vacuum-dried on the filter to remove excess water. The washed mycelia were collected from the filter paper using a spatula and wrapped in aluminum foil immediately. Under normal conditions of growth, 100 ml of culture produced approximately 1.5–2 g (wet weight) of mycelia. Approximately 0.1 g (wet weight) of mycelia were dispensed in 5 ml PYG medium containing 10 mM [3H]itraconazole (: 120 000 cpm/ml) and incubated at 35°C for various time intervals (0–120 min) for the time course study. For dose dependence study, the mycelial suspensions were incubated for 60 min at 35°C in the presence of 1.25–10 mM itraconazole. At the end of the incubation period, the mycelia were collected by filtration and washed extensively with sterile distilled water (250 ml). The washed mycelia were collected from the filter and incubated in 0.5 ml of perchloric acid (75%) at 65°C for 30 min. The mycelia were completely digested by the acid-treatment and a homogeneous suspension was obtained. The acid digested mycelial suspension was mixed thoroughly with 10 ml scintillation fluid (Aqua-
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sol, New England Nuclear, Boston, MA) and the radioactivity associated with the mycelia was determined by scintillation counting. To study the effect of the respiratory inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on the intracellular accumulation of itraconazole, 0.1 g mycelia each from the parent and the mutant isolates were incubated in the presence of 10 mM [3H]itraconazole. CCCP (final concentration 200 mM) was added after 30 min of incubation, and the intracellular accumulation of [3H]itraconazole in the mycelia before and after the addition of CCCP was measured as described above.
2.6. Antifungals Amphotericin B (batch no. 20-914-29670) was obtained from Squibb Institute for Medical Research, Princeton, NJ. Itraconazole (R51 211, batch no. STAN-9304-005-1) miconazole (R14 889, batch no. G5707), ketoconazole (R41 400, batch no. G3A 747) and [3H]itraconazole (13.5 Gbq/mmol) were from Janssen Pharmaceutica, Beerse, Belgium. The compounds were dissolved in dimethyl sulphoxide (DMSO) at a concentration of 1 mg/ml and stored as 0.25-ml aliquots at −20°C. The frozen stock was thawed at room temperature and vortexed gently several times to ensure that any crystals present were completely dissolved before use. Comparable concentrations of DMSO were used to examine its effect on the growth of A. fumigatus. No detectable inhibition of growth occurred at the concentrations used. Since amphotericin B is light-sensitive, the stock solutions and the MIC tubes were covered with aluminum foil to protect from light exposure. The following ranges of concentrations were
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used in the study: amphotericin B, 0.125–16 mg/l; itraconazole, 0.125–16 mg/l; ketoconazole, 1–128 mg/l; miconazole, 1–32 mg/l.
3. Results
3.1. Selection and susceptibility studies Approximately 2× 108 conidia were used in the initial selection of miconazole resistant mutants of A. fumigatus. In the presence of 32 mg miconazole per ml, 19 colonies were obtained on PYG agar after 6 days of incubation at 35°C, an estimated frequency of 0.95× 10 − 7. The miconazole selected isolates were then screened on PYG agar containing 16 mg itraconazole per ml. Of the 19 isolates examined, only two (MCZ14 and MCZ15) grew (frequency 1×10 − 8) in the presence of itraconazole. The in vitro susceptibilities of these two isolates to various antifungal agents are shown in Table 1. Both MCZ14 and MCZ15 had significantly higher MICs for miconazole, ketoconazole and itraconazole, but not for amphotericin B. For both isolates, the rise in MIC for itraconazole was significantly higher than that obtained for other antifungal agents. The genetic stability of itraconazole resistance was tested by repeated subculturing of the resistant isolates in the absence of the antifungal agent followed by MIC determination. No change in the MIC value for itraconazole was obtained after ten cycles of subculturing (one cycle of subculturing involves the initiation of culture from conidial suspension and allowing the culture to grow until it conidiates). The MIC of itraconazole obtained for MCZ14, but not for MCZ15, was dependent on the medium used for the susceptibility
Table 1 A comparison of the in vitro susceptibilities of itraconazole-resistant and -susceptible isolates of A. fumigatus to various antifungal agentsa A. fumigatus
Antifungal agent
MIC (mg/l) range
MIC (mg/l) mean 9S.D.
Fold MIC increase
W73355 (parent strain)
AMB ITZ MCZ KTZ AMB ITZ MCZ KTZ AMB ITZ MCZ KTZ
0.25–0.5 0.25–0.5 2 8–16 0.5 8–16 8–16 64 0.5 4–8 16 64–128
0.31 9 0.125 0.31 9 0.125 2.0 90 10 94 0.5 90 14 99 10 94 64 90 0.5 9 0 5 92 16 90 112 932
1 1 1 1 1.6 45.16 5.0 6.0 1.6 16.12 6.4 11.2
MCZ14
MCZ15
a
Each MIC value represents the range and mean of three independent MIC determinations in PYG medium. AMB, amphotericin B; ITZ, itraconazole; KTZ, ketoconazole; MCZ, miconazole. Pair-wise comparison of the MIC values of various antifungal agents for the mutants with those obtained for the parent strain by two-tailed t-test showed that all values are significant except those obtained for amphotericin B. Fold MIC increase was calculated by dividing the mean MIC value of an antifungal agent obtained for the resistant isolate with the corresponding mean MIC value obtained for the susceptible isolate.
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Fig. 1. Effect of itraconazole on the growth of susceptible and resistant A. fumigatus isolates. The experiment was repeated twice with similar results. Each point represents the mean of two independent determinations and the standard deviations were 5 10% of the mean value. The data presented above is obtained for a typical experiment. Symbols: , W73355; , MCZ14; , MCZ15.
test. This isolate grew poorly in RPMI 1640 compared to PYG broth. Consequently, the MIC obtained in RPMI 1640 after 48 h of incubation at 35°C was two to four dilutions lower than that obtained in PYG medium. However, the 72- and 96-h MIC readings showed few differences. In addition to MIC determinations, we used growth inhibition and kill curve experiments to examine the susceptibility of mutant isolates resistant to itraconazole. Since the MIC was defined as the concentration of the drug that inhibited growth of the organism within a specified time period, mutant isolates with slow growth rates in the presence of the drug may not have grown sufficiently to be detected within the specified time period. However, prolonged incubation will allow the resistant organism to grow, whereas under the same conditions the susceptible organism will fail to grow
irrespective of the incubation time. Thus, growth inhibition is a more suitable parameter for distinguishing dose dependent response of the resistant and the susceptible isolates. As shown in Fig. 1, the growth of the susceptible isolate was almost completely inhibited at 0.5 mg/l itraconazole, whereas for the resistant isolates complete inhibition was achieved at ] 4 mg/l. At 0.5 mg/l of itraconazole the wet weight of mycelia recovered for the susceptible parent was in the milligram range, whereas the growth of MCZ14 and MCZ15 was affected minimally. Although the MICs of itraconazole for MCZ14 and MCZ15 showed an approximate threefold difference (Table 1), the growth inhibition of these two isolates in the presence of various concentrations of itraconazole is significantly different. At 4 mg/l, growth of MCZ15 was inhibited ] 95% compared to the drugfree control, whereas MCZ14 showed no significant inhibition at this concentration of the drug. In contrast to its fungistatic effect on pathogenic yeast such as C. albicans, itraconazole is fungicidal agent against A. fumigatus (E.K. Manavathu and P.H. Chandrasekar, unpublished data). We therefore investigated the fungicidal activity of itraconazole against W73355, MCZ14 and MCZ15. As shown in Fig. 2, both MCZ14 and MCZ15 showed significantly reduced susceptibility to the fungicidal activity of itraconazole compared to the susceptible parent. The fungicidal activity of the drug was more dependent on the time of exposure than the concentration of the drug used.
3.2. Itraconazole uptake studies The accumulation of radiolabeled itraconazole in MCZ14 and MCZ15 was examined, and the results were compared with those obtained for the susceptible parent. The intracellular accumulation of itraconazole
Fig. 2. Fungicidal activity of itraconazole against A. fumigatus isolates susceptible (W73355) and resistant (MCZ14 and MCZ15) to itraconazole. The experiment was performed twice with similar results. Each point on the graph represents the mean of two independent determinations with the standard deviation 520% of the mean value. The data presented were obtained for a typical experiment. Itraconazole concentrations (mg/l) used: , 0; , 0.5; , 1.0; , 2.0; ", 4.0.
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Fig. 3. (A) Time dependent accumulation of [3H]itraconazole (10 mM, specific activity :1.2 × 107 cpm/mmol) in resistant (MCZ14 and MCZ15) and susceptible (W73355) isolates of A. fumigatus. (B) Concentration dependent accumulation of itraconazole for 60 min in resistant and susceptible isolates of A. fumigatus. Symbols: , W73355; , MCZ14; , MCZ15. Each experiment was repeated three times and similar results were obtained. Each point on the graph represents the mean of two independent determinations with the standard deviation 5 10% of the mean value. The results shown here were obtained for a typical experiment.
in A. fumigatus is time (Fig. 3A) and concentration (Fig. 3B) dependent; maximum level of accumulation was achieved by 30 min at 5 mM. The accumulation of itraconazole in the resistant isolates was significantly reduced compared to that in the susceptible parent. For example, the resistant isolates MCZ14 and MCZ15 accumulated approximately 60 – 80% less itraconazole in 60 min compared to the susceptible parent W73355 (Fig. 3A and B). Since the accumulation of itraconazole in A. fumigatus was a time and concentration dependent process which obeyed saturation kinetics, we examined the effect of a respiratory inhibitor carbonyl cyanide mchlorophenyl hydrazone (CCCP) to study the possible utilization of energy during the accumulation of the drug. As shown in Fig. 4, CCCP reduced the accumulation of itraconazole in the susceptible parent as well as in the resistant mutants. The significance of a CCCPdependent decrease in the intracellular accumulation of itraconazole in A. fumigatus is not understood. However, this observation suggests that the reduced intracellular accumulation of itraconazole in the resistant mutants was probably due to reduced penetration of the drug into the cells as opposed to efflux. Either mycelia or germinated conidia (but not dormant conidia) may be used for itraconazole uptake studies in A. fumigatus. In addition to using actively growing mycelia, we also used sporelings (germinated conidia) for uptake studies. The variability between replicates was significantly higher when sporelings were used perhaps due to a variety of factors. Since the dormant conidia failed to accumulate itraconazole, they had to be germinated in an appropriate growth medium for 4–6 h before uptake studies were performed. We
found it was difficult to initiate the germination of the conidia synchronously and thus the propagules were of different sizes. The high degree of variability in the size of the sporelings resulted in difficulty during scintillation counting when the filter assay technique was used. Since 3H is a weak b-particle emitter the counting efficiency was very poor without digesting the hyphal filaments. In addition, both Millipore acetate and glass fiber filters bind [3H]itraconazole resulting in a high background count. This high background count may have readily masked the small but significant differences in the accumulation of the drug.
Fig. 4. A comparison of the effect of CCCP on the accumulation of itraconazole in resistant and susceptible isolates of A. fumigatus. Symbols: , W73355; , MCZ14; , MCZ15. Each experiment was repeated three times and similar results were obtained. Each point on the graph represents the mean of two independent determinations with the standard deviation 515% of the mean value. The results shown here were obtained for a typical experiment. The arrow indicates the addition of CCCP.
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In contrast to the sporelings, the mycelia provided highly reproducible results when they were completely digested with perchloric acid. In addition, we evaluated the feasibility of using other solubilizing agents such as 6 N NaOH, 1 M HCl and certain commercial tissue solubilizers such as NCS (Amersham, Arlington, IL). None of the agents except 75% perchloric acid provided satisfactory results. Mycelia not exposed to [3H]itraconazole but digested with perchloric acid produced very low background counts (30–80 cpm) indicating that the scintillation count obtained in our experiments was not due to extraneous artifacts such as chemiluminescence as occurs when NaOH is used as a tissue solubilizer. However, it was important to cool the scintillation cocktail to ambient temperature after the addition of sample before counting to obtain stable counts.
4. Discussion Three major mechanisms for azole resistance have been reported in pathogenic yeasts such as C. albicans, Candida glabrata and Candida krusei [14]. They are: (i) reduced intracellular accumulation of the antifungal agent either due to an efflux pump or due to poor penetration of the drug across the permeability barrier; (ii) genetic modification of the target of action of azoles (cytochrome P450 dependent lanosterol 14ademethylase); and (iii) over expression of the target enzyme either by upregulation of transcription or by gene amplification. On the other hand, very little is known about the mechanisms of resistance to antifungal agents in Aspergillus species, including A. fumigatus. Paucity of clinical and environmental isolates of A. fumigatus with reduced susceptibility to azoles has handicapped investigation of the mechanisms of resistance to itraconazole in A. fumigatus in the past. In 1997, Denning et al. [8] reported the isolation and partial characterization of three clinical isolates of A. fumigatus resistant to itraconazole in vitro and in vivo using a murine disseminated aspergillosis model. Two different mechanisms were found in the three itraconazole resistant A. fumigatus isolates Denning et al. used in their study: reduced intracellular accumulation of the drug as well as possible modification of cytochrome P450 dependent 14a-demethylase as measured by the IC50s of various azoles compared to those obtained for the enzyme from the susceptible strain. The authors postulated that the reduced intracellular accumulation of the drug was possibly due to an efflux pump. Our studies on itraconazole uptake in MCZ14 and MCZ15 agree with their finding of reduced accumulation of the drug in resistant isolates. However, addition of CCCP (an inhibitor of respira-
tion) failed to increase the level of [3H]itraconazole in the cells. The fact that CCCP failed to increase the accumulation suggests that the mechanism is not mediated by an energy dependent process such as efflux. If the reduction of itraconazole accumulation in the resistant isolates was due to efflux (by pumping out the intracellularly accumulated drug at the expense of energy), addition of CCCP should have increased the amount of [3H]itraconazole accumulated in the resistant isolates, but not in the susceptible parent. This was not found; instead of an increase, a decrease in [3H]itraconazole accumulation was observed. From these results one can conclude that it is unlikely that the reduced accumulation is due to efflux. On the other hand, it is possible that the resistant isolates may have a greater permeability barrier for the entry of itraconazole than the susceptible parent. This reduced penetration may be responsible for the reduced intracellular accumulation of the drug. Both MCZ14 and MCZ15 showed cross resistance to miconazole and ketoconazole, but not to amphotericin B. This finding is consistent with our data of reduced accumulation due to poor penetration of the drug into the cells. The permeability barrier responsible for the reduced entry of itraconazole in resistant isolates also diminished the penetration of other azoles. On the other hand, penetration may not be critical for the action of amphotericin B, since its target (ergosterol) on the plasma membrane of the cell is accessible from outside without entering the cell, hence no cross resistance. Although reduced susceptibility of MCZ14 and MCZ15 to itraconazole can be explained by reduced intracellular accumulation of the drug, it is possible that the observed reduction in the susceptibility of the resistant isolates may be due to a combination of factors such as reduced penetration coupled with the modification of the target (cytochrome P450-dependent lanosterol demethylase) and increased expression of the target enzyme. In conclusion, our results suggest that under intensive selection pressure of increased antifungal usage, A. fumigatus isolates with reduced susceptibility to itraconazole may emerge and could pose a significant clinical dilemma in the management of invasive Aspergillus infections.
Acknowledgements The authors wish to thank Dr William Brown, Microbiology Laboratory, Detroit Medical Center for kindly providing the clinical isolates of various Aspergillus species used in this study. The authors also thank Dr Cornelius Janssen for kindly providing the [3H]itraconazole used in this study.
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References [1] Manavathu EK, Alangaden GJ, Lerner SA. Isolation and characterization of itraconazole-resistant mutants of Aspergillus fumigatus. Abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, 1995: American Society for Microbiology, Washington DC, USA, Abstract C, p. 570. [2] White A, Goetz MB. Azole-resistant Candida albicans: report of two cases of resistance to fluconazole and review. Clin Infect Dis 1994;19:687 – 92. [3] White TC. The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14a-demethylase in Candida albicans. Antimicrob Agents Chemother 1997;41:1488–94. [4] White TC. Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob Agents Chemother 1997;41:1482 – 7. [5] Sanglard D, Ischer F, Monod M, Bille J. Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob Agents Chemother 1995;39:2378–86. [6] Sanglard D, Ischer F, Monod M, Bille J. Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors. Antimicrob Agents Chemother 1996;40:2300–5. [7] Sanglard D, Majcherczyk P, Ischer F, Bille J. A rapid colorometric method for the study of resistance mechanism to azole
[8]
[9]
[10]
[11]
[12]
[13]
[14]
219
antifungal agents on yeasts. Clin Microbiol Infect 1997;3(Suppl 2):83. Denning DW, Venkateswarlu K, Oakley KL, et al. Itraconazole resistance in Aspergillus fumigatus. Antimicrob Agents Chemother 1997;41:1364 – 8. Oakley KL, Morrissey G, Denning DW. Efficacy of SCH-56592 in a temporarily neutropenic murine model of invasive aspergillosis with an itraconazole-susceptible and an itraconazole-resistant isolate of Aspergillus fumigatus. Antimicrob Agents Chemother 1997;41:1504 – 7. Manavathu EK, Cutright JL, Chandrasekar PH. In vitro susceptibility of itraconazole-resistant isolates of Aspergillus fumigatus to voriconazole. Clin Microbiol Infect 1997;3(suppl 2):81. Abraham OC, Manavathu EK, Chandrasekar PH. In vitro activities of voriconazole, itraconazole and amphotericin B against Aspergillus species. Abstracts of the 35th Annual Meeting of Infectious Diseases Society of America. Clin Infect Dis 1997;25:378. Manavathu EK, Alangaden G J, Lerner SA. A comparative study of the broth micro- and macro-dilution techniques for the determination of the in vitro susceptibility of Aspergillus fumigatus. Can J Microbiol 1996;42:960 – 4. Espinel-Ingroff A, Dawson K, Pfaller M, et al. Comparative and collaborative evaluation of standardization of antifungal susceptibility testing for filamentous fungi. Antimicrob Agents Chemother 1995;39:314 – 9. White TC, Marr KA, Bowden RA. Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 1998;11:382 – 402.
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Original article
Reduced susceptibility in laboratory-selected mutants of Aspergillus fumigatus to itraconazole due to decreased intracellular accumulation of the antifungal agent Elias K. Manavathu *, Jose A. Vazquez, Pranatharthi H. Chandrasekar Di6ision of Infectious Diseases, Department of Medicine, Wayne State Uni6ersity School of Medicine, Wayne State Uni6ersity, Detroit, MI 48201, USA Received 16 July 1998; accepted 6 November 1998
Abstract To study the mechanism of resistance of Aspergillus fumigatus to itraconazole, spontaneous mutants with reduced susceptibility were selected by spreading 2 ×108 conidia from a clinical isolate (W73355) susceptible to miconazole (MIC 2 mg/l), itraconazole (MIC 0.25 mg/l) and amphotericin B (MIC 0.5 mg/l) on 40 peptone yeast extract glucose agar plates containing miconazole (32 mg/l). The 19 colonies that grew (frequency 0.95 ×10 − 7) in the presence of miconazole were screened by broth macrodilution technique for their susceptibility to itraconazole. A total of two isolates (frequency 1 × 10 − 8) MCZ14 and MCZ15 had MICs of 16 and 8 mg/l, respectively, for itraconazole. Both MCZ14 and MCZ15 showed concomitant increases in MICs for ketoconazole and miconazole, but not for amphotericin B. Growth inhibition studies as well as kill curve experiments revealed that MCZ14 and MCZ15 were less susceptible to itraconazole compared to the parental strain. The intracellular accumulation of itraconazole in A. fumigatus was time and concentration dependent. Maximum accumulation was obtained within 30 min at 5 mM itraconazole. In MCZ14 and MCZ15 intracellular accumulation of [3H]itraconazole was reduced by approximately 80 and 60%, respectively, compared to the susceptible parent. The respiratory inhibitor carbonyl cyanide m-chlorophenyl hydrazone at 200 mM reduced the intracellular accumulation of itraconazole by approximately 36.2% (P 50.05) in the parent and in the mutant strains. These results suggest that (i) the reduced accumulation of itraconazole in MCZ14 and MCZ15 is due to diminished permeability of the drug and perhaps not due to efflux, (ii) the uptake of itraconazole in A. fumigatus may be an energy dependent process, and (iii) decreased accumulation of itraconazole is at least in part responsible for the reduced susceptibility of the mutant isolates to itraconazole. © 1999 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: Itraconazole; Aspergillus fumigatus; Drug resistance; Antifungal agents; Resistant mutants
1. Introduction Among various azoles approved as antifungal agents for clinical use, miconazole and itraconazole are the most effective agents against Aspergillus fumigatus. In vitro susceptibility studies have shown that itraconazole is approximately eight times more active against A. * Corresponding author. Present address: Department of Internal Medicine, 427 Lande Building, Wayne State University, 550 E. Canfield, Detroit, MI 48201, USA. Tel.: +1-313-5771931; fax: + 1313-9930302. E-mail address: [email protected] (E.K. Manavathu)
fumigatus than miconazole [1]. Although emergence of resistance to these antifungal agents has been found in pathogenic yeasts such as Candida albicans [2–7], few reports [8–10] have been published in the literature on azole resistance in A. fumigatus. Thus, the mechanism of resistance to azoles in filamentous fungi and in particular in A. fumigatus is not known. We recently screened approximately 200 clinical isolates of A. fumigatus [11] in order to obtain isolates that demonstrated reduced susceptibility to itraconazole. No A. fumigatus clinical isolate with an MIC greater than 4 mg/l for itraconazole was obtained. We therefore sought to iso-
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late A. fumigatus resistant to itraconazole in the laboratory. In this paper we describe the selection, in vitro susceptibility and studies on the accumulation of itraconazole in two mutant isolates of A. fumigatus that showed reduced susceptibility to itraconazole.
2. Materials and methods
2.1. Selection of itraconazole-resistant mutants A. fumigatus W73355, a clinical isolate obtained from the Microbiology Laboratory of the Detroit Medical Center, served as the parental strain in this investigation. A working culture of this strain was maintained on peptone yeast extract glucose (PYG: peptone 1 g; yeast extract 1 g; glucose 3 g; per liter of distilled water) agar slants at room temperature. For the preparation of conidial suspension, a culture of A. fumigatus W73355 was grown on PYG agar for 6 days at 35°C, and the conidia were collected as described previously [12]. Approximately 5×106 conidia per plate were spread on 40 PYG agar plates containing 32 mg miconazole/l. The plates were incubated for 6 days at 35°C in plastic sleeves. A total of 19 colonies that grew on PYG agar in the presence of miconazole were subcultured as individual isolates and subsequently screened on PYG agar containing itraconazole (16 mg/l) for their susceptibility. Of the 19 miconazole selected isolates, only MCZ14 and MCZ15 grew in the presence of itraconazole on PYG agar and were used for subsequent studies.
2.2. MIC determination The in vitro susceptibility of the parent and the mutant isolates of A. fumigatus to various antifungals was examined by determining the MIC of each compound using a previously described broth macrodilution technique [12,13] in both RPMI 1640 and PYG broth. Briefly, fresh conidia were resuspended in growth medium at a density of 2×104 conidia per ml. Then two times the required concentrations of the drugs were prepared in PYG or RPMI 1640 medium (0.5 ml) by serial dilution in sterile 6-ml polystyrene tubes (Falcon 2054) and inoculated with an equal volume (0.5 ml) of the conidial suspension prepared in the same medium. The tubes were incubated at 35°C for 48 h and scored for visible growth after vortexing the tubes gently, or scraping the walls of the tube followed by vortexing. The MIC was defined as the lowest concentration of the drug in which no visible growth occurred. The MIC determination for each isolate was repeated three times, and the data were within 91 dilution.
2.3. Growth inhibition study Cultures of W73355, MCZ14 and MCZ15 were grown in 100 ml PYG medium from conidia (1 ×104/ ml) in the presence of 0–4 mg/l itraconazole at 35°C for 48 h with gentle agitation (90 rpm) on an orbital shaker. The mycelia were collected on Whatman no. 2 filter paper and washed once with 250 ml sterile distilled water. The washed mycelia were harvested by vacuum-filtration and the wet weight was determined. The wet weight of the mycelia was plotted against the concentrations of itraconazole used for the susceptible and the resistant isolates to examine growth inhibition.
2.4. Kill cur6es In the presence of various concentrations of itraconazole (0–4 mg/l), 5-ml aliquots of a conidial suspension prepared in PYG broth (1× 106 conidia/ml) were incubated at 25°C. At various time intervals 0.1-ml aliquots of the conidial suspension were removed, diluted 102 – 104 fold, and 0.1-ml quantities were spread in duplicate on PYG agar plates. The plates were incubated at 35°C for 48 h and the number of colony forming units (c.f.u.) per ml of conidial suspension were calculated and plotted against the time of exposure to itraconazole for the construction of the kill curve.
2.5. Itraconazole uptake studies A. fumigatus cultures were grown from conidia (1× 104 conidia/ml) in 100 ml of PYG broth at 35°C for 48 h on a gyratory shaker (170 rpm). The mycelia were harvested by filtration using a Buchner funnel on Whatman no. 2 filter paper, washed with sterile distilled water (500 ml) and gently vacuum-dried on the filter to remove excess water. The washed mycelia were collected from the filter paper using a spatula and wrapped in aluminum foil immediately. Under normal conditions of growth, 100 ml of culture produced approximately 1.5–2 g (wet weight) of mycelia. Approximately 0.1 g (wet weight) of mycelia were dispensed in 5 ml PYG medium containing 10 mM [3H]itraconazole (: 120 000 cpm/ml) and incubated at 35°C for various time intervals (0–120 min) for the time course study. For dose dependence study, the mycelial suspensions were incubated for 60 min at 35°C in the presence of 1.25–10 mM itraconazole. At the end of the incubation period, the mycelia were collected by filtration and washed extensively with sterile distilled water (250 ml). The washed mycelia were collected from the filter and incubated in 0.5 ml of perchloric acid (75%) at 65°C for 30 min. The mycelia were completely digested by the acid-treatment and a homogeneous suspension was obtained. The acid digested mycelial suspension was mixed thoroughly with 10 ml scintillation fluid (Aqua-
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sol, New England Nuclear, Boston, MA) and the radioactivity associated with the mycelia was determined by scintillation counting. To study the effect of the respiratory inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on the intracellular accumulation of itraconazole, 0.1 g mycelia each from the parent and the mutant isolates were incubated in the presence of 10 mM [3H]itraconazole. CCCP (final concentration 200 mM) was added after 30 min of incubation, and the intracellular accumulation of [3H]itraconazole in the mycelia before and after the addition of CCCP was measured as described above.
2.6. Antifungals Amphotericin B (batch no. 20-914-29670) was obtained from Squibb Institute for Medical Research, Princeton, NJ. Itraconazole (R51 211, batch no. STAN-9304-005-1) miconazole (R14 889, batch no. G5707), ketoconazole (R41 400, batch no. G3A 747) and [3H]itraconazole (13.5 Gbq/mmol) were from Janssen Pharmaceutica, Beerse, Belgium. The compounds were dissolved in dimethyl sulphoxide (DMSO) at a concentration of 1 mg/ml and stored as 0.25-ml aliquots at −20°C. The frozen stock was thawed at room temperature and vortexed gently several times to ensure that any crystals present were completely dissolved before use. Comparable concentrations of DMSO were used to examine its effect on the growth of A. fumigatus. No detectable inhibition of growth occurred at the concentrations used. Since amphotericin B is light-sensitive, the stock solutions and the MIC tubes were covered with aluminum foil to protect from light exposure. The following ranges of concentrations were
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used in the study: amphotericin B, 0.125–16 mg/l; itraconazole, 0.125–16 mg/l; ketoconazole, 1–128 mg/l; miconazole, 1–32 mg/l.
3. Results
3.1. Selection and susceptibility studies Approximately 2× 108 conidia were used in the initial selection of miconazole resistant mutants of A. fumigatus. In the presence of 32 mg miconazole per ml, 19 colonies were obtained on PYG agar after 6 days of incubation at 35°C, an estimated frequency of 0.95× 10 − 7. The miconazole selected isolates were then screened on PYG agar containing 16 mg itraconazole per ml. Of the 19 isolates examined, only two (MCZ14 and MCZ15) grew (frequency 1×10 − 8) in the presence of itraconazole. The in vitro susceptibilities of these two isolates to various antifungal agents are shown in Table 1. Both MCZ14 and MCZ15 had significantly higher MICs for miconazole, ketoconazole and itraconazole, but not for amphotericin B. For both isolates, the rise in MIC for itraconazole was significantly higher than that obtained for other antifungal agents. The genetic stability of itraconazole resistance was tested by repeated subculturing of the resistant isolates in the absence of the antifungal agent followed by MIC determination. No change in the MIC value for itraconazole was obtained after ten cycles of subculturing (one cycle of subculturing involves the initiation of culture from conidial suspension and allowing the culture to grow until it conidiates). The MIC of itraconazole obtained for MCZ14, but not for MCZ15, was dependent on the medium used for the susceptibility
Table 1 A comparison of the in vitro susceptibilities of itraconazole-resistant and -susceptible isolates of A. fumigatus to various antifungal agentsa A. fumigatus
Antifungal agent
MIC (mg/l) range
MIC (mg/l) mean 9S.D.
Fold MIC increase
W73355 (parent strain)
AMB ITZ MCZ KTZ AMB ITZ MCZ KTZ AMB ITZ MCZ KTZ
0.25–0.5 0.25–0.5 2 8–16 0.5 8–16 8–16 64 0.5 4–8 16 64–128
0.31 9 0.125 0.31 9 0.125 2.0 90 10 94 0.5 90 14 99 10 94 64 90 0.5 9 0 5 92 16 90 112 932
1 1 1 1 1.6 45.16 5.0 6.0 1.6 16.12 6.4 11.2
MCZ14
MCZ15
a
Each MIC value represents the range and mean of three independent MIC determinations in PYG medium. AMB, amphotericin B; ITZ, itraconazole; KTZ, ketoconazole; MCZ, miconazole. Pair-wise comparison of the MIC values of various antifungal agents for the mutants with those obtained for the parent strain by two-tailed t-test showed that all values are significant except those obtained for amphotericin B. Fold MIC increase was calculated by dividing the mean MIC value of an antifungal agent obtained for the resistant isolate with the corresponding mean MIC value obtained for the susceptible isolate.
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Fig. 1. Effect of itraconazole on the growth of susceptible and resistant A. fumigatus isolates. The experiment was repeated twice with similar results. Each point represents the mean of two independent determinations and the standard deviations were 5 10% of the mean value. The data presented above is obtained for a typical experiment. Symbols: , W73355; , MCZ14; , MCZ15.
test. This isolate grew poorly in RPMI 1640 compared to PYG broth. Consequently, the MIC obtained in RPMI 1640 after 48 h of incubation at 35°C was two to four dilutions lower than that obtained in PYG medium. However, the 72- and 96-h MIC readings showed few differences. In addition to MIC determinations, we used growth inhibition and kill curve experiments to examine the susceptibility of mutant isolates resistant to itraconazole. Since the MIC was defined as the concentration of the drug that inhibited growth of the organism within a specified time period, mutant isolates with slow growth rates in the presence of the drug may not have grown sufficiently to be detected within the specified time period. However, prolonged incubation will allow the resistant organism to grow, whereas under the same conditions the susceptible organism will fail to grow
irrespective of the incubation time. Thus, growth inhibition is a more suitable parameter for distinguishing dose dependent response of the resistant and the susceptible isolates. As shown in Fig. 1, the growth of the susceptible isolate was almost completely inhibited at 0.5 mg/l itraconazole, whereas for the resistant isolates complete inhibition was achieved at ] 4 mg/l. At 0.5 mg/l of itraconazole the wet weight of mycelia recovered for the susceptible parent was in the milligram range, whereas the growth of MCZ14 and MCZ15 was affected minimally. Although the MICs of itraconazole for MCZ14 and MCZ15 showed an approximate threefold difference (Table 1), the growth inhibition of these two isolates in the presence of various concentrations of itraconazole is significantly different. At 4 mg/l, growth of MCZ15 was inhibited ] 95% compared to the drugfree control, whereas MCZ14 showed no significant inhibition at this concentration of the drug. In contrast to its fungistatic effect on pathogenic yeast such as C. albicans, itraconazole is fungicidal agent against A. fumigatus (E.K. Manavathu and P.H. Chandrasekar, unpublished data). We therefore investigated the fungicidal activity of itraconazole against W73355, MCZ14 and MCZ15. As shown in Fig. 2, both MCZ14 and MCZ15 showed significantly reduced susceptibility to the fungicidal activity of itraconazole compared to the susceptible parent. The fungicidal activity of the drug was more dependent on the time of exposure than the concentration of the drug used.
3.2. Itraconazole uptake studies The accumulation of radiolabeled itraconazole in MCZ14 and MCZ15 was examined, and the results were compared with those obtained for the susceptible parent. The intracellular accumulation of itraconazole
Fig. 2. Fungicidal activity of itraconazole against A. fumigatus isolates susceptible (W73355) and resistant (MCZ14 and MCZ15) to itraconazole. The experiment was performed twice with similar results. Each point on the graph represents the mean of two independent determinations with the standard deviation 520% of the mean value. The data presented were obtained for a typical experiment. Itraconazole concentrations (mg/l) used: , 0; , 0.5; , 1.0; , 2.0; ", 4.0.
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Fig. 3. (A) Time dependent accumulation of [3H]itraconazole (10 mM, specific activity :1.2 × 107 cpm/mmol) in resistant (MCZ14 and MCZ15) and susceptible (W73355) isolates of A. fumigatus. (B) Concentration dependent accumulation of itraconazole for 60 min in resistant and susceptible isolates of A. fumigatus. Symbols: , W73355; , MCZ14; , MCZ15. Each experiment was repeated three times and similar results were obtained. Each point on the graph represents the mean of two independent determinations with the standard deviation 5 10% of the mean value. The results shown here were obtained for a typical experiment.
in A. fumigatus is time (Fig. 3A) and concentration (Fig. 3B) dependent; maximum level of accumulation was achieved by 30 min at 5 mM. The accumulation of itraconazole in the resistant isolates was significantly reduced compared to that in the susceptible parent. For example, the resistant isolates MCZ14 and MCZ15 accumulated approximately 60 – 80% less itraconazole in 60 min compared to the susceptible parent W73355 (Fig. 3A and B). Since the accumulation of itraconazole in A. fumigatus was a time and concentration dependent process which obeyed saturation kinetics, we examined the effect of a respiratory inhibitor carbonyl cyanide mchlorophenyl hydrazone (CCCP) to study the possible utilization of energy during the accumulation of the drug. As shown in Fig. 4, CCCP reduced the accumulation of itraconazole in the susceptible parent as well as in the resistant mutants. The significance of a CCCPdependent decrease in the intracellular accumulation of itraconazole in A. fumigatus is not understood. However, this observation suggests that the reduced intracellular accumulation of itraconazole in the resistant mutants was probably due to reduced penetration of the drug into the cells as opposed to efflux. Either mycelia or germinated conidia (but not dormant conidia) may be used for itraconazole uptake studies in A. fumigatus. In addition to using actively growing mycelia, we also used sporelings (germinated conidia) for uptake studies. The variability between replicates was significantly higher when sporelings were used perhaps due to a variety of factors. Since the dormant conidia failed to accumulate itraconazole, they had to be germinated in an appropriate growth medium for 4–6 h before uptake studies were performed. We
found it was difficult to initiate the germination of the conidia synchronously and thus the propagules were of different sizes. The high degree of variability in the size of the sporelings resulted in difficulty during scintillation counting when the filter assay technique was used. Since 3H is a weak b-particle emitter the counting efficiency was very poor without digesting the hyphal filaments. In addition, both Millipore acetate and glass fiber filters bind [3H]itraconazole resulting in a high background count. This high background count may have readily masked the small but significant differences in the accumulation of the drug.
Fig. 4. A comparison of the effect of CCCP on the accumulation of itraconazole in resistant and susceptible isolates of A. fumigatus. Symbols: , W73355; , MCZ14; , MCZ15. Each experiment was repeated three times and similar results were obtained. Each point on the graph represents the mean of two independent determinations with the standard deviation 515% of the mean value. The results shown here were obtained for a typical experiment. The arrow indicates the addition of CCCP.
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In contrast to the sporelings, the mycelia provided highly reproducible results when they were completely digested with perchloric acid. In addition, we evaluated the feasibility of using other solubilizing agents such as 6 N NaOH, 1 M HCl and certain commercial tissue solubilizers such as NCS (Amersham, Arlington, IL). None of the agents except 75% perchloric acid provided satisfactory results. Mycelia not exposed to [3H]itraconazole but digested with perchloric acid produced very low background counts (30–80 cpm) indicating that the scintillation count obtained in our experiments was not due to extraneous artifacts such as chemiluminescence as occurs when NaOH is used as a tissue solubilizer. However, it was important to cool the scintillation cocktail to ambient temperature after the addition of sample before counting to obtain stable counts.
4. Discussion Three major mechanisms for azole resistance have been reported in pathogenic yeasts such as C. albicans, Candida glabrata and Candida krusei [14]. They are: (i) reduced intracellular accumulation of the antifungal agent either due to an efflux pump or due to poor penetration of the drug across the permeability barrier; (ii) genetic modification of the target of action of azoles (cytochrome P450 dependent lanosterol 14ademethylase); and (iii) over expression of the target enzyme either by upregulation of transcription or by gene amplification. On the other hand, very little is known about the mechanisms of resistance to antifungal agents in Aspergillus species, including A. fumigatus. Paucity of clinical and environmental isolates of A. fumigatus with reduced susceptibility to azoles has handicapped investigation of the mechanisms of resistance to itraconazole in A. fumigatus in the past. In 1997, Denning et al. [8] reported the isolation and partial characterization of three clinical isolates of A. fumigatus resistant to itraconazole in vitro and in vivo using a murine disseminated aspergillosis model. Two different mechanisms were found in the three itraconazole resistant A. fumigatus isolates Denning et al. used in their study: reduced intracellular accumulation of the drug as well as possible modification of cytochrome P450 dependent 14a-demethylase as measured by the IC50s of various azoles compared to those obtained for the enzyme from the susceptible strain. The authors postulated that the reduced intracellular accumulation of the drug was possibly due to an efflux pump. Our studies on itraconazole uptake in MCZ14 and MCZ15 agree with their finding of reduced accumulation of the drug in resistant isolates. However, addition of CCCP (an inhibitor of respira-
tion) failed to increase the level of [3H]itraconazole in the cells. The fact that CCCP failed to increase the accumulation suggests that the mechanism is not mediated by an energy dependent process such as efflux. If the reduction of itraconazole accumulation in the resistant isolates was due to efflux (by pumping out the intracellularly accumulated drug at the expense of energy), addition of CCCP should have increased the amount of [3H]itraconazole accumulated in the resistant isolates, but not in the susceptible parent. This was not found; instead of an increase, a decrease in [3H]itraconazole accumulation was observed. From these results one can conclude that it is unlikely that the reduced accumulation is due to efflux. On the other hand, it is possible that the resistant isolates may have a greater permeability barrier for the entry of itraconazole than the susceptible parent. This reduced penetration may be responsible for the reduced intracellular accumulation of the drug. Both MCZ14 and MCZ15 showed cross resistance to miconazole and ketoconazole, but not to amphotericin B. This finding is consistent with our data of reduced accumulation due to poor penetration of the drug into the cells. The permeability barrier responsible for the reduced entry of itraconazole in resistant isolates also diminished the penetration of other azoles. On the other hand, penetration may not be critical for the action of amphotericin B, since its target (ergosterol) on the plasma membrane of the cell is accessible from outside without entering the cell, hence no cross resistance. Although reduced susceptibility of MCZ14 and MCZ15 to itraconazole can be explained by reduced intracellular accumulation of the drug, it is possible that the observed reduction in the susceptibility of the resistant isolates may be due to a combination of factors such as reduced penetration coupled with the modification of the target (cytochrome P450-dependent lanosterol demethylase) and increased expression of the target enzyme. In conclusion, our results suggest that under intensive selection pressure of increased antifungal usage, A. fumigatus isolates with reduced susceptibility to itraconazole may emerge and could pose a significant clinical dilemma in the management of invasive Aspergillus infections.
Acknowledgements The authors wish to thank Dr William Brown, Microbiology Laboratory, Detroit Medical Center for kindly providing the clinical isolates of various Aspergillus species used in this study. The authors also thank Dr Cornelius Janssen for kindly providing the [3H]itraconazole used in this study.
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References [1] Manavathu EK, Alangaden GJ, Lerner SA. Isolation and characterization of itraconazole-resistant mutants of Aspergillus fumigatus. Abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, 1995: American Society for Microbiology, Washington DC, USA, Abstract C, p. 570. [2] White A, Goetz MB. Azole-resistant Candida albicans: report of two cases of resistance to fluconazole and review. Clin Infect Dis 1994;19:687 – 92. [3] White TC. The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14a-demethylase in Candida albicans. Antimicrob Agents Chemother 1997;41:1488–94. [4] White TC. Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob Agents Chemother 1997;41:1482 – 7. [5] Sanglard D, Ischer F, Monod M, Bille J. Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob Agents Chemother 1995;39:2378–86. [6] Sanglard D, Ischer F, Monod M, Bille J. Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors. Antimicrob Agents Chemother 1996;40:2300–5. [7] Sanglard D, Majcherczyk P, Ischer F, Bille J. A rapid colorometric method for the study of resistance mechanism to azole
[8]
[9]
[10]
[11]
[12]
[13]
[14]
219
antifungal agents on yeasts. Clin Microbiol Infect 1997;3(Suppl 2):83. Denning DW, Venkateswarlu K, Oakley KL, et al. Itraconazole resistance in Aspergillus fumigatus. Antimicrob Agents Chemother 1997;41:1364 – 8. Oakley KL, Morrissey G, Denning DW. Efficacy of SCH-56592 in a temporarily neutropenic murine model of invasive aspergillosis with an itraconazole-susceptible and an itraconazole-resistant isolate of Aspergillus fumigatus. Antimicrob Agents Chemother 1997;41:1504 – 7. Manavathu EK, Cutright JL, Chandrasekar PH. In vitro susceptibility of itraconazole-resistant isolates of Aspergillus fumigatus to voriconazole. Clin Microbiol Infect 1997;3(suppl 2):81. Abraham OC, Manavathu EK, Chandrasekar PH. In vitro activities of voriconazole, itraconazole and amphotericin B against Aspergillus species. Abstracts of the 35th Annual Meeting of Infectious Diseases Society of America. Clin Infect Dis 1997;25:378. Manavathu EK, Alangaden G J, Lerner SA. A comparative study of the broth micro- and macro-dilution techniques for the determination of the in vitro susceptibility of Aspergillus fumigatus. Can J Microbiol 1996;42:960 – 4. Espinel-Ingroff A, Dawson K, Pfaller M, et al. Comparative and collaborative evaluation of standardization of antifungal susceptibility testing for filamentous fungi. Antimicrob Agents Chemother 1995;39:314 – 9. White TC, Marr KA, Bowden RA. Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 1998;11:382 – 402.
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