JAC Advance Access originally published online on January 24, 2008
Journal of Antimicrobial Chemotherapy 2008 61(4):798-804; doi:10.1093/jac/dkn015
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Original research |
Susceptibility of clinical isolates of Candida species to fluconazole and detection of Candida albicans ERG11 mutations
Department of Dermatology, Qilu Hospital, Shandong University, Ji’nan 250012, China
* Corresponding author. Tel: +86-53182169390; Fax: +86-53186927544; E-mail: drlichunyang{at}hotmail.com
Received 1 October 2007; returned 23 October 2007; revised 18 November 2007; accepted 31 December 2007
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Abstract |
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Objectives: To investigate fluconazole susceptibility of Candida albicans from non-AIDS patients and to analyse the relationship between mutations in the ERG11 gene of these isolates and fluconazole resistance.
Methods: Four hundred and twenty-six clinical isolates of Candida species were collected. Fluconazole susceptibility was tested in vitro using microdilution and disc diffusion assays. The ERG11 genes of 23 isolates of C. albicans (8 susceptible and 15 resistant) and 6 type strains (4 susceptible and 2 resistant) were amplified in three overlapping regions of the gene and sequenced.
Results: Of the 426 isolates collected, 68.6% were C. albicans; however, only 5.1% were resistant to fluconazole. Eighteen silent mutations and 19 missense mutations were detected. There were six missense mutations only in resistant isolates or resistant type strains: (i) G487T (A114S) and T916C (Y257H) appeared simultaneously in 14 fluconazole-resistant isolates without any other mutation; these may be associated with resistance; (ii) T541C (Y132H) and T1559C (I471T) are known to contribute to fluconazole resistance; and (iii) C1567A (Q474K) or a novel mutation T1493A (F449Y) was identified but correlations with resistance have not been studied.
Conclusions: In this survey, C. albicans is the major cause of candidiasis in non-AIDS patients, and some isolates that are resistant to fluconazole have G487T and T916C mutations in ERG11 that are associated with fluconazole resistance.
Keywords:
C. albicans
,
drug susceptibility testing
,
lanosterol 14
-demethylase
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Introduction |
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Candida albicans is a commensal and important opportunistic human pathogen causing common ailments such as thrush, vaginitis and invasive disease in immunocompromised patients. Repeated azole therapy for chronic infections, especially in AIDS patients, has been associated with an increase in azole resistance.1 It is necessary for clinicians to understand the antifungal susceptibility profiles of Candida species before treatment is initiated. A prospective surveillance for candidaemia at 16 hospitals in Iowa, USA, revealed that C. albicans was the most important cause of candidaemia, and 3% of Candida species were resistant to fluconazole.2 Many studies have identified azole-resistant strains of C. albicans from AIDS patients, whereas we focused on those isolates from non-AIDS candidiasis patients, with the idea of identifying other mutations in ERG11 that contribute to azole resistance.
Thus far, there are at least four explanations of azole resistance in C. albicans. The first is based on spatial configuration changes of the target enzyme 14-demethylase (Erg11p) due to mutations in the encoding gene ERG11. Erg11p is a key enzyme in the ergosterol synthesis pathway of C. albicans. Ergosterol is essential for maintaining the integrity and function of C. albicans membrane. Erg11p is a member of the cytochrome P450 superfamily. The protein is composed of 528 amino acids that form 13 -helices of A to M, several β-pleated sheets and some other helix configurations such as A', J', K' and K''. The active centre of Erg11p is located deep inside the protein, near the haemachrome between helices I and L. The substrate interacts with a long access channel and is then demethylated.3–6 Azoles block this process and inhibit ergosterol synthesis. ERG11 contains 1851 bp. The transcription start codon is located at 148–150 bp and the stop codon at 1732–1734 bp (referring to a published ERG11 sequence in GenBank, accession no. X13296
[GenBank]
). If one or more mutations in ERG11 result in changes in the Erg11p spatial configuration, a decrease in the affinity between the azole and protein occurs. This altered phenotype often makes isolates resistant to azole. In addition, overexpression of ERG11 has been thought to increase resistance, although recent data indicate that overexpression is unrelated to azole resistance in C. albicans.7,8
Amino acid substitutions have been described in 61 residues of the Erg11p due to missense mutations in ERG11 gene, as shown in Table 1. Erg11p mutations Y132H, T315A, S405F, G464S, R467K and I471T have been shown to cause azole resistance.5,9–13 The following mutations may exist in susceptible strains and are not thought able to confer resistance, such as F72L, K99T, F105L, D116E, K128T, G129A, K147R, D153E, E266D, E266Q, K287R, G303D, L305P, K342R, V437I and D446N.5,6,8,12,14–17 Some mutations can exert an effect only when combined with other mutations. For example, the G129A and G464S mutations result in relative increases in the MICs of azole derivatives compared with strains with only the G464S mutation.5 For other mutations, their contribution to resistance is uncertain, such as Y79C, D81G, F126L, V130I, Y132F, K143R, K143E, F145L, A149V, E165Y, T199I, T229A, P230L, I253V, R265G, R267H, D278E, D278N, S279F, H283D, D294G, G307S, M374V, F380S, P386L, E391G, H400R, V404L, N440K, G448E, G448V, G448R, F449L, F449S, G450E, V452A, G465S, F487L, V488G, T494P, P503L and D504G.6,8,13,15–21
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Multidrug transporters also contribute to drug resistance in Candida species. Overexpression of Candida drug resistance genes CDR1 and CDR2 as well as the multiple drug resistance gene MDR1 can decrease the intracellular drug concentration effectively.8,24 Also, biofilms may mediate antifungal resistance. The C. albicans biofilm like that of other species is a highly heterogeneous bilayered structure, composed of cellular and non-cellular elements and a matrix consisting of carbohydrate, protein and other components.25–27 Biofilms represent a niche for microorganisms where they are thought to be protected from the host immune system and antimicrobial therapies. Finally, fluconazole sequestration within intracellular vacuoles may be a novel mechanism of resistance.28
Multiple mechanisms of fluconazole resistance can arise in a single C. albicans isolate,16 and there also are chances that even a single base change in ERG11 can increase resistance to azoles.9,10 We amplified and sequenced the ERG11 gene of C. albicans isolates in order to identify mutations that might be related to fluconazole resistance.
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Isolates and type strains
Clinical isolates of Candida species were collected from patients with vulvovaginal candidiasis (VVC), mucocutaneous candidiasis, candidal infection of respiratory, urinary and digestive tracts or from patients with candidaemia. The following type strains were obtained from the CCCCM (Chinese Cultural Collection Commission for Microbiology, Nanjing, China): fluconazole-susceptible C. albicans (genotype A) C1b, C1c, C1d and C1e, and fluconazole-resistant C. albicans (genotype A) ATCC 76615-19, Candida dubliniensis C8a, Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019. The Darlington strain of C. albicans (genotype A) was provided by Professor John E. Bennett of National Institutes of Health, Bethesda, MD, USA.
Modified Sabouraud dextrose agar (SDA) contained 20 g of dextrose, 10 g of peptone, 20 g of agar, 50 mg of chloromycetin and distilled water to 1000 mL. Triphenyltetrazolium chloride (TTC) agar was composed of SDA with 0.005% TTC. Corn meal–Tween agar (CMA) contained 40 g of corn meal, 10 mL of Tween 80, 15 g of agar and distilled water to 1000 mL. Beef extract agar (3 g of beef extract, 5 g of NaCl, 10 g of peptone, 25 g of agar and distilled water to 1000 mL) and carbon assimilation medium [5 g of (NH4)2SO4, 1 g of KH2PO4, 0.5 g of MgSO4·7H2O, 0.2 g of yeast extract, 20 g of agar and distilled water to 1000 mL] were also used. All the media except TTC were autoclaved at 121°C for 15 min. CHROMagar was prepared as recommended by the manufacturer. RPMI 1640 broth [10.4 g of RPMI 1640 powder (with glutamine but without NaHCO3), 34.53 g of MOPS and distilled water to 1000 mL, pH 6.9–7] was prepared and sterilized by filtration. RPMI 1640 agar medium was composed of RPMI 1640 broth with 2% dextrose and 1.5% agar.
Fluconazole (FCZ20603001, purity 99.9%) was obtained from Qilu Pharmaceutical Factory, while fluconazole tablets (ROSCO, Denmark) and the yeast DNA kit (OMEGA, USA) were also used.
Isolates collection and identification
Clinical specimens were inoculated on modified SDA slants, incubated, isolated and maintained at 4°C. Candida species were identified by TTC reduction, germ tube formation in serum at 37°C, chlamydospore formation on CMA at 25°C, sugar assimilations, CHROMagar and the Vitek 2 system (bioMérieux, France).
Fluconazole susceptibility tests
Susceptibility and resistance of strains were measured using the M27-A2 broth dilution method, as recommended by the CLSI, and by disc diffusion assays. Strains ATCC 6258 and ATCC 22019 were used as controls.
Genotyping of C. albicans and identification of C. dubliniensis
Genomic DNA of C. albicans isolates was obtained following the manufacturer’s instructions using the yeast DNA kit. After extraction, the concentration of DNA solution was adjusted to 0.01 g/L. Primer pairs CA-INT-L (5'-ATAAGGGAAGTCGGCAAAATAGATCCGTAA-3') and CA-INT-R (5'-CCTTGGCTGTGGTTTCGCTAGATAGTAGAT-3') were used to amplify a DNA fragment that spans the site of the transposable intron in the 25S rDNA.29 Amplification was in a 25 µL volume containing 2.5 µL of 10x buffer (with Mg2+), 1 µL of 0.01 g/L genomic DNA, 0.5 µL of 10 mM dNTP, 0.5 µL of 50 pmol/µL each primer and 0.25 µL of 5 U/µL Taq polymerase. PCR conditions were as follows: 94°C for 3 min, 94°C for 1 min, 65°C for 1 min and 72°C for 2.5 min, for 33 cycles; and 72°C for 10 min. PCRs resulted in a single product for C. albicans genotypes A (450 bp) and B (840 bp) and C. dubliniensis (1080 bp). But C. albicans genotype C isolates yielded two PCR products (450 and 840 bp). All the isolates identified as C. albicans or C. dubliniensis by routine methods and VITEK 2 system were genotyped, with C1b, C1c, C1d, C1e and C8a as controls.
Amplifying ERG11 gene and sequencing
Three pairs of primers were designed: ERGSec1A (5'-TTAGTGTTTTATTGGATTCCTTGGTT-3') and ERGSec1B (5'-TCTCATTTCATCACCAAATAAAGATC-3') yielded an expected PCR product extending from 295 to 777 bp of the ERG11 gene; ERGSec2A (5'-ACCAGAAATTACTATTTTCACTGCTTCA-3') and ERGSec2B (5'-AAGTCAAATCATTCAAATCACCA CCT-3') yielded an expected PCR product extending from 723 to 1204 bp of the ERG11 gene; and ERGSec3A (5'-AGGTGGTGATTTGAATGATTTGACTT-3') and ERGSec3B (5'-GAACTATAATCAGGGTCAGGCACTTT-3') gave an expected PCR product extending from 1179 to 1667 bp of the ERG11 gene. For all of the reactions, a 25 µL volume contained 2.5 µL of 10x PCR buffer (with Mg2+), 1 µL of 0.01 g/L genomic DNA, 0.5 µL of 10 mM dNTP, 0.5 µL of 50 pmol/µL each primer and 0.25 µL of 5 U/µL Pfu polymerase. PCR conditions were as follows: 94°C for 5 min; 94°C for 1 min, 61°C for 1 min and 72°C for 1 min, for 33 cycles; and 72°C for 7 min. The products were purified and sequenced with an ABI PRISM 3700 DNA Analyzer (Applied Biosystems). The ERG11 of all the resistant isolates, eight susceptible isolates and the six type strains were amplified and sequenced. Two strains whose ERG11 sequence is known (ATCC 76615-19 and the Darlington strain) were used as controls.11,19
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Identification of clinical isolates of Candida spp
A total of 426 Candida isolates were collected, in which 263 isolates were from VVC patients, 101 isolates from sputa, 22 isolates from urine, 16 isolates from skin, 7 isolates from blood, 6 isolates from mouth and 11 isolates from other sites (Table 2). C. albicans (68.8%), Candida tropicalis (12.2%) and Candida glabrata (10.8%) were represented among the isolates. On CHROMagar plates, colonies of 219 isolates were light green and 79 isolates were dark green. Data from the VITEK 2 system confirmed that the 219 isolates that were light green on CHROMagar were C. albicans, but only 4 of the 79 dark green isolates were identified as C. dubliniensis, and the other 75 isolates were identified as C. albicans. Four isolates of C. dubliniensis identified by VITEK 2 and one isolate that was dark green on CHROMagar but identified as C. albicans by VITEK 2 was confirmed to be C. dubliniensis by electrophoresis of PCR-amplified fragments of 25S rDNA. Among the 293 isolates of C. albicans, 203 isolates were genotype A (PCR product of 450 bp), while 89 isolates were genotype B (PCR product of 840 bp) and 1 isolate was genotype C (PCR products of 450 and 840 bp).
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Fluconazole susceptibility
Fifteen isolates of C. albicans were resistant to fluconazole, including 3 isolates (592, 4263 and 4266) from different patients in Ji'nan, Eastern China, and 12 isolates (GZ03, GZ04, GZ09, GZ15, GZ16, GZ17, GZ18, GZ23, GZ29, GZ34, GZ51 and GZ58) from different patients in Guangzhou, Southern China. All of the 15 resistant isolates were C. albicans genotype A. Among 52 isolates of C. tropicalis, only 1 was resistant, while there were 9 resistant isolates of 46 isolates of C. glabrata.
The three sections of ERG11 sequence amplified by PCR were 483, 482 and 489 bp in length, respectively. Each PCR product of all the C. albicans isolates resulted in a clear band on ethidium bromide-stained, UV-transilluminated agarose gel. The sequenced ERG11 gene was 1454 bp in length. Thirty-seven mutations were found in 29 C. albicans isolates or strains by sequencing their ERG11 PCR products, including 18 silent mutations and 19 missense mutations. Eight missense mutations were identified in the resistant isolates and 13 missense mutations were identified in the susceptible isolates, as shown in Tables 3 and 4.
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G487T and T916C were present simultaneously in the following 14 resistant isolates: 592, 4263, 4266, GZ03, GZ09, GZ15, GZ16, GZ17, GZ18, GZ23, GZ29, GZ34, GZ51 and GZ58. These mutations were absent in other ERG11 mutations. In another resistant isolate GZ04, four missense mutations T495A, A530C, T541C and T1493A were detected. In the sequences of susceptible type strains, homozygous T495A, A504C, G820T and A945C and heterozygous T495A/T, T566G/T, G630T/G, G635C/G, C1271A/C and G1289T/G were found. No missense mutation was detected in the susceptible isolates C261, 0461 or 2855. Homozygous T495A, A530C, G640A, G820C and A945C and heterozygous A945C/A and G1609A/G were found in the other five susceptible isolates.
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C. albicans and C. dubliniensis are similar phenotypically when routine lab methods are used that are based on their morphological, physiological or biochemical characteristics. The green colour of a C. dubliniensis colony on CHROMagar medium is usually darker than that of C. albicans, but this difference will disappear upon prolonged culture or in stored isolates.30 Results of C. albicans and C. dubliniensis by VITEK 2 system also could be incorrect.31 Similarly, in our study, not all of the isolates of C. dubliniensis were identified correctly by VITEK 2. We amplified the sequence of the 25S rDNA transposable intron in order to differentiate these two species and subtype C. albicans isolates. C. albicans genotype B has an intron in the 25S rDNA, whereas genotype A does not. C. dubliniensis also has an intron in the same location, but it is larger than that in C. albicans genotype B. PCR with primers CA-INT-L/R resulted in a single product for C. albicans genotypes A (450 bp) and B (840 bp) and C. dubliniensis (1080 bp), while the C. albicans genotype C has two major products (450 and 840 bp).29
Among the 426 isolates, most were vaginal or from sputa and only 62 isolates were from urine, blood, skin, mouth or other sites. C. albicans is a prevalent fungal pathogen in candidiasis. About half of infections were caused by C. albicans. It is even more common in patients with VVC.32 Nearly two-thirds (263/426) of the isolates in our study were from VVC patients, of which C. albicans accounted for 66.5% (175/263), but still lower in frequency than isolates from sputa (78/101). A total 68.8% of all isolates were identified as C. albicans, while C. tropicalis (12.2%) and C. glabrata (10.8%) were the other most common species.
Both microdilution and disc diffusion methods are recommended by CLSI in fluconazole susceptibility testing of C. albicans. MIC can be read directly with the quantitative microdilution method, but tailing of C. albicans in this format makes the determinations of breakpoints difficult to read. The disc diffusion method is easier to do, but it is qualitative, cannot provide MIC values and the results are variable when the moisture capacity of the medium is changed by evaporation.
C. albicans isolates from immunocompromised patients usually experience a lower antifungal susceptibility.33 They may have changed their phenotype under drug selection pressure during a long-term antifungal therapy. Fluconazole resistance of such isolates may reach 35%.34 As for the candidal isolates from other patients, susceptibility to fluconazole may be as high as 95.8%.33,35 In our study, fluconazole resistance of Candida species and C. albicans was 8.5% and 5.1%, respectively. It should be noted that 15.6% (41/263) of VVC cases were caused by C. glabrata and the fluconazole resistance ratio of this species was 19.6%. This situation might create problems in the clinical treatment of some VVC cases if an antifungal susceptibility test cannot be carried out in advance.
The 295–1667 bp sequence of ERG11 includes all known missense mutations listed in Table 1. The C- and N-termini of Erg11p are on the surface of the protein and not at the active site. Therefore, we amplified the 295–1667 bp part of ERG11 gene to look for possible mutations.
The mutations detected in the control strains (ATCC 76615-19 and the Darlington strain), shown in Table 3, were similar to reported sequences.11,19 These 15 resistant isolates were from different patients in two different cities of China, but their ERG11 sequence appeared completely identical except for GZ04. No other mutations were detected in the 14 isolates except G487T (A114S) and T916C (Y257H), which are different from most other reports in regard to the accordance of so many isolates. There is the possibility that the 14 isolates are derived from the same resistant strain in the population. But recently, mutations G487T and T916C were found in an induced fluconazole-resistant strain of C. albicans along with T395A (F83Y), and neither of them was present in the fluconazole-susceptible parent strain or any other susceptible strain.22 These data imply that G487T and/or T916C might be correlated with fluconazole resistance in C. albicans. A114S is near the substrate channel in Erg11p and may change the affinity between Erg11p and an azole more than Y257H, which is located in the G helix, far away from the active centre or substrate access channel of the protein. However, there is no experimental proof of this until this mutant is constructed and its antifungal susceptibility is tested. Furthermore, other resistance mechanisms need to be measured in these strains such as overexpression of MDR1 and CDR1/2.
Four mutations were detected in the ERG11 of GZ04. Homozygous T541C (Y132H) contributes to azole resistance;11 while T495A (D116E) and A530C (K128T) do not.5,14 The fourth T1493A (F449Y) is a novel mutation. It has been reported that T1492C and T1493C in ERG11 of resistant isolates resulted in F449L and F449S of Erg11p.6,15 F449 is close to the terminal of the I helix and located in front of the haem group. Substitution may influence the function of the active centre. But different mutations at this position might affect the enzyme in other ways; some mutations might render the enzyme resistant and other mutations may have no effect or even render the cell more susceptible. The role of the three substitutions of F449 in susceptibility/resistance cannot be defined until other experimental data are provided.
Among the 13 missense mutations in these strains, A945C (E266D) was frequently observed in isolates reported previously, just like T495A (D116E), but this mutation cannot cause fluconazole resistance.14 Position V488 is far away from the active centre of Erg11p and therefore the G1609A (V488I) mutation may not result in fluconazole resistance.6 Previously, other research groups found that V488I was present in fluconazole-resistant but not fluconazole-susceptible strains, a result that indicates a correlation with the azole-resistant phenotype.12,14,36 Furthermore, Maebashi et al.12 reported that V488I might alter the affinity between the drug and target in the enzyme–drug molecule complex. However, we found a heterozygous G1609A in ERG11 in the susceptible strain 02928. It implies that G1609A (V488I) in C. albicans does not contribute to the formation of fluconazole resistance. Or, the allele with the mutation G1609A might not be transcribed and the non-mutated allele transcribed, because heterozygosity in genes of C. albicans may imply that one of the two alleles is not transcribed.
Since E165 is close to the active centre of the target enzyme, perhaps the mutation E165Y might be associated with fluconazole and itraconazole resistance.6 Because E165K was detected in fluconazole-susceptible isolate 02928, the E165 position seems less important for fluconazole resistance and E165K cannot result in fluconazole resistance, although E165Y might influence Erg11p configuration differently. Two kinds of mutations at position D225 were detected in our study, G820T (D225Y) in type strain C1c and G820C (D225H) in isolate C522. Theoretically, D225 is located in the F helix of Erg11p, far away from the I helix or the active centre, and substitutions of D225 cannot influence the affinity between fluconazole and the target. Also, both C1c and C522 were susceptible to fluconazole; thus, these two mutations are not related to fluconazole resistance.
There was one homozygous and five heterozygous missense mutations in C1e. Although K161N and R163T are much closer to the active centre than K119N, M140R, P375Q or R381I, they do not confer fluconazole resistance because strain C1e was susceptible.
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This study was supported by a grant from the National Science Foundation of China (No. 30471563).
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None to declare.
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Acknowledgements |
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We thank Professor Xi Liyan (Sun Yat-sen University, China), Professor Li Ruoyu (Peking University, China), Professor Shen Yongnian (Chinese Cultural Collection Commission for Microbiology, China) and Professor John E. Bennett (National Institutes of Health, USA) for the supply of isolates or type strains. We also thank Professor Richard A. Calderone (Georgetown University, USA) for reading through the paper and editorial suggestions.
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1 Cowen LE, Sanglard D, Calabrese D, et al. Evolution of drug resistance in experimental populations of Candida albicans. J Bacteriol (2000) 182:1515–22.
2
Diekema DJ, Messer SA, Brueggemann AB, et al. Epidemiology of candidemia: 3-year results from the emerging infections and the epidemiology of Iowa organisms study. J Clin Microbiol (2002) 40:1298–302.
3
Ji H, Zhang W, Zhou Y, et al. A three-dimensional model of lanosterol 14-demethylase of Candida albicans and its interaction with azole antifungals. J Med Chem (2000) 43:2493–505.[CrossRef][Web of Science][Medline]
4
Podust LM, Poulos TL, Waterman MR. Crystal structure of cytochrome P450 14-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors. Proc Natl Acad Sci USA (2001) 98:3068–73.
5
Sanglard D, Ischer F, Koymans L, et al. Amino acid substitutions in the cytochrome P-450 lanosterol 14-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents. Antimicrob Agents Chemother (1998) 42:241–53.
6
Marichal P, Koymans L, Willemsens S, et al. Contribution of mutations in the cytochrome P450 14-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans. Microbiology (1999) 10:2701–13.
7
White TC. The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14 demethylase in Candida albicans. Antimicrob Agents Chemother (1997) 41:1488–94.[Abstract]
8
White TC, Holleman S, Dy F, et al. Resistance mechanisms in clinical isolates of Candida albicans. Antimicrob Agents Chemother (2002) 46:1704–13.
9
Lamb DC, Kelly DE, Schunck WH, et al. The mutation T315A in Candida albicans sterol 14-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity. J Biol Chem (1997) 272:5682–8.
10
Lamb DC, Kelly DE, White TC, et al. The R467K amino acid substitution in Candida albicans sterol 14-demethylase causes drug resistance through reduced affinity. Antimicrob Agents Chemother (2000) 44:63–7.
11
Kakeya H, Miyazaki Y, Miyazaki H, et al. Genetic analysis of azole resistance in the Darlington strain of Candida albicans. Antimicrob Agents Chemother (2000) 44:2985–90.
12 Maebashi K, Kudoh M, Nishiyama Y, et al. 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. Microbiol Immunol (2003) 47:117–24.[Web of Science][Medline]
13 Kamai Y, Maebashi K, Kudoh M, et al. Characterization of mechanisms of fluconazole resistance in a Candida albicans isolate from a Japanese patient with chronic mucocutaneous candidiasis. Microbiol Immunol (2004) 48:937–43.[Web of Science][Medline]
14 Löffler J, Kelly SL, Hebart H, et al. Molecular analysis of cyp51 from fluconazole resistant Candida albicans strains. FEMS Microbiol Lett (1997) 151:263–8.[Web of Science][Medline]
15
Perea S, López-Ribot JL, Kirkpatrick WR, et al. Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients. Antimicrob Agents Chemother (2001) 45:2676–84.
16 Goldman GH, da Silva Ferreira ME, dos Reis Marques E, et al. Evaluation of fluconazole resistance mechanisms in Candida albicans clinical isolates from HIV-infected patients in Brazil. Diagn Microbiol Infect Dis (2004) 50:25–32.[CrossRef][Web of Science][Medline]
17 Wang YB, Wang H, Guo HY, et al. Analysis of ERG11 gene mutation in Candida albicans. Di Yi Jun Yi Da Xue Bao (2005) 25:1390–3.
18
Li X, Brown N, Chau AS, et al. Changes in susceptibility to posaconazole in clinical isolates of Candida albicans. J Antimicrob Chemother (2004) 53:74–80.
19
Long F, Zhang YX, Lan HK, et al. The point mutation of cytochrome P-450 lanosterol 14- demethylase ERG11 gene in fluconazole-resistant Candida albicans. Chin J Infect Dis (2002) 20:211–4.
20
Asai K, Tsuchimori N, Okonogi K, et al. Formation of azole-resistant Candida albicans by mutation of sterol 14-demethylase P450. Antimicrob Agents Chemother (1999) 43:1163–9.
21 Wang WL, Wang DL, Li RY, et al. A study of the resistant mechanisms of Candida albicans to azhole antifungal agents. Chin J Derm Venereol (1999) 13:3–5.
22 Jiang WS, Tan SS, Jiang GY, et al. Synergistic effect of terbinafine combined with fluconazole or itraconazole on stable fluconazole-resistant Candida albicans induced by fluconazole in vitro. Chin J Microbiol Immunol (2006) 26:360–4.
23
Marr KA, Lyons CN, Rustad TR, et al. Rapid, transient fluconazole resistance in Candida albicans is associated with increased mRNA levels of CDR. Antimicrob Agents Chemother (1998) 42:2584–9.
24 Park S, Perlin DS. Establishing surrogate markers for fluconazole resistance in Candida albicans. Microb Drug Resist (2005) 11:232–8.[CrossRef][Web of Science][Medline]
25
Andes D, Nett J, Oschel P, et al. Development and characterization of an in vivo central venous catheter Candida albicans biofilm model. Infect Immun (2004) 72:6023–31.
26
Chandra J, Kuhn DM, Mukherjee PK, et al. Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J Bacteriol (2001) 183:5385–94.
27
Baillie GS, Douglas LJ. Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents. J Antimicrob Chemother (2000) 46:397–403.
28 Maebashi K, Kudoh M, Nishiyama Y, et al. A novel mechanism of fluconazole resistance associated with fluconazole sequestration in Candida albicans isolates from a myelofibrosis patient. Microbiol Immunol (2002) 46:317–26.[Web of Science][Medline]
29
McCullough MJ, Clemons KV, Stevens DA, et al. Molecular and phenotypic characterization of genotypic Candida albicans subgroups and comparison with Candida dubliniensis and Candida stellatoidea. J Clin Microbiol (1999) 37:417–21.
30 Schoofs A, Odds FC, Colebunders R, et al. Use of specialised isolation media for recognition and identification of Candida dubliniensis isolates from HIV-infected patients. Eur J Clin Microbiol Infect Dis (1997) 16:296–300.[CrossRef][Web of Science][Medline]
31 Cárdenes-Perera CD, Torres-Lana A, Alonso-Vargas R, et al. Evaluation of API ID 32C and VITEK-2 to identify Candida dubliniensis. Diagn Microbiol Infect Dis (2004) 50:219–21.[CrossRef][Web of Science][Medline]
32 De Vos MM, Cuenca-Estrella M, Boekhout T, et al. Vulvovaginal candidiasis in a Flemish patient population. Clin Microbiol Infect (2005) 11:1005–11.[CrossRef][Web of Science][Medline]
33
St-Germain G, Laverdiere M, Pelletier R, et al. Prevalence and antifungal susceptibility of 442 Candida isolates from blood and other normally sterile sites: results of a 2-year (1996 to 1998) multicenter surveillance study in Quebec, Canada. J Clin Microbiol (2001) 39:949–53.
34 Akbar DH, Tahawi AT. Candidemia at a university hospital: epidemiology, risk factors and predictors of mortality. Ann Saudi Med (2001) 21:178–82.[Web of Science][Medline]
35 Bedini A, Venturelli C, Mussini C, et al. Epidemiology of candidaemia and antifungal susceptibility patterns in an Italian tertiary-care hospital. Clin Microbiol Infect (2006) 12:75–80.[CrossRef][Web of Science][Medline]
36 Cernicka J, Subik J. Resistance mechanisms in fluconazole-resistant Candida albicans isolates from vaginal candidiasis. Int J Antimicrob Agents (2006) 27:403–8.[CrossRef][Web of Science][Medline]
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