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Genetic basis for differential activities of fluconazole and voriconazole against Candida krusei.

Fukuoka T, Johnston DA, Winslow CA, de Groot MJ, Burt C, Hitchcock CA, Filler SG.

Division of Infectious Diseases, Department of Medicine, Harbor-UCLA Research and Education Institute, Torrance, California 90502, USA.

Invasive infections caused by Candida krusei are a significant concern because this organism is intrinsically resistant to fluconazole. Voriconazole is more active than fluconazole against C. krusei in vitro. One mechanism of fluconazole resistance in C. krusei is diminished sensitivity of the target enzyme, cytochrome P450 sterol 14alpha-demethylase (CYP51), to inhibition by this drug. We investigated the interactions of fluconazole and voriconazole with the CYP51s of C. krusei (ckCYP51) and fluconazole-susceptible Candida albicans (caCYP51). We found that voriconazole was a more potent inhibitor of both ckCYP51 and caCYP51 in cell extracts than was fluconazole. Also, the ckCYP51 was less sensitive to inhibition by both drugs than was caCYP51. These results were confirmed by expressing the CYP51 genes from C. krusei and C. albicans in Saccharomyces cerevisiae and determining the susceptibility of the transformants to voriconazole and fluconazole. We constructed homology models of the CYP51s of C. albicans and C. krusei based on the crystal structure of CYP51 from Mycobacterium tuberculosis. These models predicted that voriconazole is a more potent inhibitor of both caCYP51 and ckCYP51 than is fluconazole, because the extra methyl group of voriconazole results in a stronger hydrophobic interaction with the aromatic amino acids in the substrate binding site and more extensive filling of this site. Although there are multiple differences in the predicted amino acid sequence of caCYP51 and ckCYP51, the models of the two enzymes were quite similar and the mechanism for the relative resistance of ckCYP51 to the azoles was not apparent.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12654649&dopt=Abstract fluconazole Diflucan



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Flucytosine-fluconazole cross-resistance in purine-cytosine permease-deficient Candida lusitaniae clinical isolates: indirect evidence of a fluconazole uptake transporter.

Noel T, Francois F, Paumard P, Chastin C, Brethes D, Villard J.

Laboratoire des Sciences Vegetales, Faculte de Pharmacie, Universite Rene Descartes-Paris 5, Paris 75006, USA. noel pharmacie.univ-paris5.fr

An unusual interaction between flucytosine and fluconazole was observed when a collection of 60 Candida lusitaniae clinical isolates was screened for cross-resistance. Among eight isolates resistant to flucytosine (MIC >/= 128 micro g/ml) and susceptible to fluconazole (0.5 < MIC < 2 micro g/ml), four became flucytosine-fluconazole cross resistant when both antifungals were used simultaneously. Fluconazole resistance occurred only in the presence of high flucytosine concentrations, and the higher the fluconazole concentration used, the greater the flucytosine concentration necessary to trigger the cross-resistance. When the flucytosine- and fluconazole-resistant cells were grown in the presence of fluconazole alone, the cells reversed to fluconazole susceptibility. Genetic analyses of the progeny from crosses between resistant and sensitive isolates showed that resistance to flucytosine was derived from a recessive mutation in a single gene, whereas cross-resistance to fluconazole seemed to vary like a quantitative trait. We further demonstrated that the four clinical isolates were susceptible to 5-fluorouracil and that cytosine deaminase activity was unaffected. Kinetic transport studies with [(14)C]flucytosine showed that flucytosine resistance was due to a defect in the purine-cytosine permease. Our hypothesis was that extracellular flucytosine would subsequently behave as a competitive inhibitor of fluconazole uptake transport. Finally, in vitro selection of spontaneous and induced mutants indicated that such a cross-resistance mechanism could also affect other Candida species, including C. albicans, C. tropicalis, and C. glabrata. This is the first report of a putative fluconazole uptake transporter in Candida species and of a possible resistance mechanism associated with a deficiency in the uptake of this drug.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12654658&dopt=Abstract fluconazole Diflucan



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In vitro and in vivo activity of tea tree oil against azole-susceptible and -resistant human pathogenic yeasts.

Mondello F, De Bernardis F, Girolamo A, Salvatore G, Cassone A.

Laboratory of Bacteriology and Medical Mycology, Istituto Superiore di Sanita, Rome, Italy. mondello iss.it

A tea tree oil (TTO) preparation of defined chemical composition was studied, using a microbroth method, for its in vitro activity against 115 isolates of Candida albicans, other Candida species and Cryptococcus neoformans. The fungal strains were from HIV-seropositive subjects, or from an established type collection, including reference and quality control strains. Fourteen strains of C. albicans resistant to fluconazole and/or itraconazole were also assessed. The same preparation was also tested in an experimental vaginal infection using fluconazole-itraconazole-susceptible or -resistant strains of C. albicans. TTO was shown to be active in vitro against all tested strains, with MICs ranging from 0.03% (for C. neoformans) to 0.25% (for some strains of C. albicans and other Candida species). Fluconazole- and/or itraconazole-resistant C. albicans isolates had TTO MIC50s and MIC90s of 0.25% and 0.5%, respectively. TTO was highly efficacious in accelerating C. albicans clearance from experimentally infected rat vagina. Three post-challenge doses of TTO (5%) brought about resolution of infection regardless of whether the infecting C. albicans strain was susceptible or resistant to fluconazole. Overall, the use of a reliable animal model of infection has confirmed and extended our data on the therapeutic effectiveness of TTO against fungi, in particular against C. albicans.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12668571&dopt=Abstract fluconazole Diflucan



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Evaluation of semisolid agar screening tests for determining fluconazole and amphotericin B susceptibilities of Candida strains by using three different media.

Esen N, Yucesoy M, Yulug N.

Dokuz Eylul University, School of Medicine, Department of Microbiology and Clinical Microbiology, Izmir, Turkey. nuran.esen deu.edu.tr

The susceptibilities of 164 Candida isolates against fluconazole and amphotericin B were determined by semisolid agar screening tests and the microdilution method according to NCCLS M27-A standards. The semisolid agar screening tests were performed with three different media containing 0.5% agar and 2, 8, and 40 microg/ml of fluconazole or 0.5 and 2.0 microg/ml of amphotericin B. These media were MOPS buffered RPMI 1640, brain-heart infusion and 1/3 diluted Sabouraud dextrose agar. The results of both methods were interpreted as susceptible, dose dependent susceptible and resistant for fluconazole and susceptible and resistant for amphotericin B. The agreement rates of semisolid agar screening tests using RPMI 1640, brain-heart infusion and Sabouraud dextrose media with the reference microdilution method were found to be 71.4%, 51.2%, and 57.3% for fluconazole and 79.3%, 53.7%, and 56.7% for amphotericin B, respectively. Overall, we conclude that semisolid agar screening tests using RPMI 1640 can be used for determining the susceptibilities of Candida isolates against fluconazole and amphotericin B in clinical microbiology laboratories.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12678412&dopt=Abstract fluconazole Diflucan



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Proliferation of intracellular structure corresponding to reduced affinity of fluconazole for cytochrome P-450 in two low-susceptibility strains of Candida albicans isolated from a Japanese AIDS patient.

Maebashi K, Kudoh M, Nishiyama Y, Makimura K, Kamai Y, Uchida K, Yamaguchi H.

Teikyo University Institute of Medical Mycology, Hachioji, Tokyo 192-0395, Japan. kazunori_maebashi meiji.co.jp

Three Candida albicans isolates, TIMM 3164, 3165 and 3166 with reduced fluconazole susceptibility, were isolated from two Japanese AIDS patients. We earlier reported that a reduced intracellular accumulation of fluconazole in these isolates played an important role in the resistance mechanism of fluconazole, but we did not exclude the involvement of other factors. We here examined characteristics related to cytochrome P-450 (CYP), especially sterol 14alpha-demethylase encoded by the ERG11 gene which is the target molecule for fluconazole. In TIMM 3164 and 3165, the ergosterol synthesis by cell-free extracts was somewhat less susceptible to fluconazole, due to a decrease in fluconazole affinity for CYP. The nucleotide substitutions in the ERG11 gene were identified to result in three amino acid changes of K143R, E266D and V488I in TIMM 3164, and of E266D, V404L and V488I in TIMM 3165. These amino acid substitutions might contribute to the decreased affinity for CYP in both isolates. However, a single amino acid change, E266D, observed in TIMM 3166 was unrelated to the decreased affinity for CYP. The most prominent finding on the ultrastructure of TIMM 3164 and 3165 was the development of mesh membrane structures of the endoplasmic reticula, which is a location related to sterol synthesis. This phenomenon was not observed in the cells of TIMM 3166 or the susceptible control strains of ATCC 90028 and 10231. In addition to the reduced intracellular accumulation, the decreased affinity of fluconazole for CYP in TIMM 3164 and 3165 is assumed to be associated with the fluconazole-resistance phenotype.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12680714&dopt=Abstract fluconazole Diflucan



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Activities of fluconazole and voriconazole against 1,586 recent clinical isolates of Candida species determined by Broth microdilution, disk diffusion, and Etest methods: report from the ARTEMIS Global Antifungal Susceptibility Program, 2001.

Pfaller MA, Diekema DJ, Messer SA, Boyken L, Hollis RJ.

Department of Pathology, Roy J. and Lucille A. Carver College of Medicine and College of Public Health, University of Iowa, Iowa City, Iowa 52242, USA. michael-pfaller uiowa.edu

The ARTEMIS Global Antifungal Susceptibility Program (ARTEMIS Program) was initiated in 2001 to provide focused surveillance of the activities of fluconazole and voriconazole against Candida spp. isolated from blood and other normally sterile sites. A total of 1,586 episodes of infection were detected at 61 international study sites. Overall, 57.7% of the infections were due to Candida albicans, followed by C. glabrata (14.8%), C. parapsilosis (12.5%), C. tropicalis (9.4%), C. krusei (2.7%), and C. lusitaniae (1.5%). Isolates of C. albicans, C. parapsilosis, and C. tropicalis were all highly susceptible to fluconazole (for 99% of the isolates the MICs were <or=8 microg/ml). Likewise, 99 to 100% of these species were inhibited by <or=1 microg of voriconazole per ml. Voriconazole was also active against C. glabrata (93% of the isolates were susceptible [MICs <or= 1 microg/ml]) and C. krusei (100% of the isolates were susceptible). The agar-based Etest and disk diffusion methods performed well for the testing of both fluconazole and voriconazole compared to the broth microdilution MIC reference method. These observations establish the continued importance of C. albicans as a pathogen and the sustained activity of fluconazole and the broad spectrum of activity of voriconazole and will serve as the first-year benchmark for the ARTEMIS Program. Continued surveillance and refinement of broth- and agar-based test methods will help to identify susceptibility trends and improve the laboratory capability for antifungal susceptibility testing.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12682127&dopt=Abstract fluconazole Diflucan



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Liquid/liquid extraction using 96-well plate format in conjunction with hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the analysis of fluconazole in human plasma.

Eerkes A, Shou WZ, Naidong W.

Covance Laboratories Inc, Department of Bioanalytical Chemistry, 3301 Kinsman Boulevard, Madison, WI 53704, USA.

A bioanalytical method using automated sample transferring, automated liquid/liquid extraction (LLE) and hydrophilic interaction liquid chromatography-tandem mass spectrometry was developed for the determination of fluconazole in human plasma. Samples of 0.05 ml were transferred into 96-well plate using automatic liquid handler (Multiprobe II). Automated LLE was carried out on a 96-channel programmable liquid handling workstation (Quadra 96) using methyl-tetra butyl ether as the extraction solvent. The extract was evaporated to dryness, reconstituted, and injected onto a silica column using an aqueous-organic mobile phase. The chromatographic run time was 2.0 min per injection, with retention times of 1.47 and 1.44 min for fluconazole and internal standard (IS) ritonavir, respectively. The detection was by monitoring fluconazole at m/z 307-->238 and IS at m/z 721-->296, respectively. The standard curve range was 0.5-100 ng ml(-1). The inter-day precision and accuracy of the quality control samples were <7.1% relative standard deviation and <2.2% relative error. Copyright 2003 Elsevier Science B.V.

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Mode of selection and experimental evolution of antifungal drug resistance in Saccharomyces cerevisiae.

Anderson JB, Sirjusingh C, Parsons AB, Boone C, Wickens C, Cowen LE, Kohn LM.

Department of Botany, University of Toronto, Mississauga, Ontario L5L 1C6, Canada.

We show that mode of selection, degree of dominance of mutations, and ploidy are determining factors in the evolution of resistance to the antifungal drug fluconazole in yeast. In experiment 1, yeast populations were subjected to a stepwise increase in fluconazole concentration over 400 generations. Under this regimen, two mutations in the same two chromosomal regions rose to high frequency in parallel in three replicate populations. These mutations were semidominant and additive in their effect on resistance. The first of these mutations mapped to PDR1 and resulted in the overexpression of the ABC transporter genes PDR5 and SNQ2. These mutations had an unexpected pleiotropic effect of reducing the residual ability of the wild type to reproduce at the highest concentrations of fluconazole. In experiment 2, yeast populations were subjected to a single high concentration of fluconazole. Under this regimen, a single recessive mutation appeared in each of three replicate populations. In a genome-wide screen of approximately 4700 viable deletion strains, 13 were classified as resistant to fluconazole (ERG3, ERG6, YMR102C, YMR099C, YPL056C, ERG28, OSH1, SCS2, CKA2, SML1, YBR147W, YGR283C, and YLR407W). The mutations in experiment 2 all mapped to ERG3 and resulted in the overexpression of the gene encoding the drug target ERG11, but not PDR5 and SNQ2. Diploid hybrids from experiments 1 and 2 were less fit than the parents in the presence of fluconazole. In a variation of experiment 2, haploids showed a higher frequency of resistance than diploids, suggesting that degree of dominance and ploidy are important factors in the evolution of antifungal drug resistance.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12702675&dopt=Abstract fluconazole Diflucan