Changes in Genotype and Fluconazole Susceptibility of Isolates from Patients with Candida glabrata in Tunisia

Thérapie 2014 Septembre-Octobre; 69 (5): 449–455 DOI: 10.2515/therapie/2014059

PHARMACOGENETICS

© 2014 Société Française de Pharmacologie et de Thérapeutique

Changes in Genotype and Fluconazole Susceptibility of Isolates from Patients with Candida glabrata in Tunisia Salma Abbes1, Imen Amouri1, Hayet Sellami1, Sourour Neji1, Houaida Trabelsi1, Fatma Cheikhrouhou1, Fattouma Makni1, Stéphane Ranque2 and Ali Ayadi1 1 Laboratoire de biologie moléculaire, parasitaire et fongique, Faculté de médecine, Université de Sfax, Tunisie 2 Laboratoire de parasitologie-mycologie, Aix-Marseille Université, AP-HM, CHU Timone, Marseille, France Text received October 7th, 2013; accepted April 1st, 2014

Keywords: Candida glabrata; fluconazole; gene expression; microsatellites analysis

Abstract – Candida glabrata has emerged as an opportunistic pathogen of considerable importance in invasive and superficial infections. Aims. To analyze the development of fluconazole resistance in patients under treatment through epidemiological survey in our hospital. Patients and methods. Twenty two patients (89 clinical strains) were collected. Molecular typing of isolates was performed by polymorphic markers. Analysis of gene expression was realized by reverse transcriptase-real time polymerase chain reactions (RT-qPCR). Results. Genetic analysis showed that 63% persists with apparently unchanged strains (n=14). Among them, four showed fluconazole resistance development. A strain replacement was observed in 6 patients and two patients selected more resistant isolates during the course of treatment. An analysis of Candida glabrata cerebellar degeneration-related protein 1 (CgCDR1), Candida glabrata cerebellar degeneration-related protein 2 (CgCDR2) and Candida glabrata sterol 14 alpha-demetylase Erg 11 (CgERG11) expression revealed an over-expression in 10 resistant isolates. Conclusion. This study demonstrated that C. glabrata strain undergo frequent changes in vivo. The increase in CgCDR1 and CgCDR2 expression was the most mechanism associated with fluconazole resistance.

Mots clés : Candida glabrata ; fluconazole ; expression de gènes ; analyse microsatellites

Résumé – Changement de génotype et de sensibilité au fluconazole de souches de Candida glabrata en Tunisie. Candida glabrata a émergé comme un pathogène opportuniste d’une importance considérable dans les infections invasives et superficielles. Objectifs. Etudier le développement de résistance au fluconazole et ses mécanismes chez des patients sous traitement antifongique. Patients et méthodes. Vingt-deux patients (89 souches cliniques) ont été suivis. Le typage moléculaire des isolats a été réalisé par quatre marqueurs polymorphes. L’analyse de l’expression des gènes a été réalisée par reverse transcriptase-real time polymerase chain reactions (RT-qPCR). Résultats. Pour 63 % des patients, les souches isolées étaient génotypiquement identiques (n = 14). Parmi eux, quatre ont développé une résistance au fluconazole. Un remplacement de souche a été observé chez 6 patients. Parmi eux, deux patients ont présenté une sélection d’isolats résistants. Une analyse de l’expression des gènes Candida glabrata cerebellar degeneration-related protein 1 (CgCDR1), Candida glabrata cerebellar degeneration-related protein 2 (CgCDR2) et Candida glabrata sterol 14 alpha-demetylase Erg 11 (CgERG11) a révélé une surexpression chez 10 isolats résistants. Conclusion. Cette étude a démontré que les populations de C. glabrata sont soumises à des fréquents changements in vivo. L’augmentation de l’expression des gènes CgCDR1 et CgCDR2 semble être associée à la résistance au fluconazole.

Abbreviations: see end of article.

1. Introduction Candida glabrata has emerged as a major pathogen, causing mucosal and systemic infections.[1,2] In many countries, a high

frequency of Candida glabrata isolates showed a high fluconazole resistance rate.[3] Candida glabrata is of concern because it is less susceptible to azole antifungal agents when compared with other Candida spp.[4,5] A clear understanding of the epidemiology of

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Candida glabrata infection requires a reliable typing system for the evaluation of isolate relatedness. Microsatellite or variable tandem repeat are more commonly used for Candida glabrata when it’s provided a high discriminatory power.[6-9] Although, the mechanisms implicated in fluconazole resistance are still under search, increasing level transcript in Candida glabrata cerebellar degeneration-related protein 1 (CgCDR1) and Candida glabrata cerebellar degeneration-related protein 2 (CgCDR2) responsible for drug efflux were in most studies associated with fluconazole resistance in C. glabrata isolates and deletion of these genes resulted in hypersensitivity to azoles.[10-16] Our aims were to describe the development of fluconazole resistance in patients under treatment through an epidemiological survey of fluconazole resistance in our hospital. A change in CgCDR1, CgCDR2 and Candida glabrata sterol 14 alpha-demethylase ERG 11 (CgERG11) genes expression was also estimated to search their contribution in fluconazole resistance.

2. Materials and methods 2.1. Patients and isolates The population of Candida glabrata clinical isolates included in this present study was recovered during an epidemiological survey of antifungal resistance conducted at the Habib Bourguiba University Hospital in Sfax from January 2005 to December 2007 as described.[6] Twenty two patients were re-assessed and sequential isolates were collected from each patient. The same patients and isolates that were previously studied to approve new microsatellite markers,[6] were included in our study for gene expression analyses. Eighty nine strains were isolated from peripheral site from consecutive episodes of urinary or vaginal infection. Isolates were collected from inpatients in different wards (intensive care unit, infectious disease unit, nephrology or endocrinology unit) and from outpatients. Sequential samples were collected with an interval time between the first and the latest one varying from one week to several months for each patient. Isolates were plated onto Candiselect 4 medium (BioRad, France) at 37 °C for 48h and then identification of Candida glabrata was realized by ID32C (Biomérieux, France) assimilation test and Glabrata RTT kit (Fumouze, Diagnostic, France). 2.2. Susceptibility testing Susceptibility to fluconazole was determined by measuring the minimum inhibitory concentration (MIC) obtained through the use of the E-test® (Biomérieux, France), a routine susceptibility testing method in use in our laboratory and adapted from the clinical and laboratory standards institute (CLSI) reference method.[17] In this assay, a pure fresh culture on yeast extract peptone dextrose (YEPD) agar plates was suspended in 2 mL of 0.85M NaCl to match a 0.5

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McFarland standard. The inoculum was swabbed into RPMI 1640 agar plates (AES laboratories, Cambourg, France) and allowed to dry for 15 min before E-test® strips were applied. Plates were incubated at 37 °C and MIC was read after 24h and 48h as the drug concentration at which the inhibition ellipse intercepted the scale on the antifungal strips. A MIC ≥64 µg/mL defined fluconazole resistance.[17] 2.3. Ribonucleotidic acid and deoxyribonucleic acid isolation Total ribonucleotidic acid (RNA) was extracted from yeasts cells picked in the logarithmic phase of growth at 48 hours of incubation with an RNAeasy protect mini kit (Qiagen, France), according to the instructions of the manufacturer. Elution was done at 70 °C. RNA extracts were treated by RNase-free DNase (Qiagen, France) to avoid deoxyribonucleic acid (DNA)/RNA contamination. Genomic DNA was extracted with MasterPureTM yeast DNA purification kit (Epicentre Biotechnologies, Madison USA) method. 2.4. Candida glabrata typing The Candida glabrata isolates were typed using four markers, three microsatellites (MTI, GLM4 and GLM5) and irregular patterns in the C-5 sterol desaturase (ERG3), as previously described.[6,7] In our study, we have considered that there is an acquisition of resistance if isolates from the same patient were identical (all studied loci are identical) and a selection if they were genetically different (different by more than one loci).

2.5. Reverse transcriptase-PCR RNA extract of each isolate was copied into cDNA by reverse transcriptase (RT)-PCR, performed with a 25 µL volume with 2.5 µL of MuLva buffer (Biology, France), 2U of MuLva transcriptase reverse (Biology), 5 µL of each RNA sample, 0.5 µM of mixture of random primers and polyA (Eurogentec, France). Samples were subjected to reverse transcription at 37 °C for 60 min.

2.6. Real-time PCR Quantitative real-time PCR (qPCR) analysis was performed to measure the expression level of each of the three target genes, CgCDR1, CgCDR2 (which encode efflux pumps) and CgERG11 (which encodes the azole target enzyme). The amount of each gene’s transcript was normalized to URA3, a single copy gene encoding orotidine 5-phosphate decarboxylase. Two µl cDNA template were used to amplify each of the four genes, CgCDR1, CgCDR2 and CgERG11, in separate qPCR

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reactions as previously described.[14] Briefly, 200 pM of each primer, 200 pM of hydrolysis probes and 25 µL of Taq Man mix (Applied Biosystems, Courtaboeuf, France) were used. Amplification consisted in an initial step at 94 °C for 10 min, and then 40 cycles of 15s at 94 °C, 30s at 55 °C and 20s at 72 °C. Fluorescence data were collected during the extension step and analysed with a MX4000 (Stratagen, France) real-time PCR machine. Each reaction was run in duplicate and the mean of two values was calculated. The amplification efficiency was determined for each gene. The normalized relative quantities or the gene-specific product in each sample was calculated. This means that the quantities of the samples of interest were compared to a calibrator (reference strain with high susceptibility to fluconazole [TU10], a fluconazole susceptible control [MIC=0.125 mg/L]). The quantities of both the calibrator and the samples of interest were normalized to URA3.[18] For qualitative analysis, we chose a threshold value of 2.5 normalized gene expression to define gene over-expression.

3. Results Susceptibility testing: a total of 89 Candida glabrata isolates from consecutive infections were screened for fluconazole resistance by E-test. In our study, most of colonizing strains isolated (90%) were initially susceptible (MIC≤8µg/mL). In only two patients (n°5 and 8), isolates were initially resistant (MIC≥64 µg/ mL) [table I]. From analyzed patients, six patients developed fluconazole resistance as shown in table I. Molecular typing of Candida glabrata isolates and gene expression analysis: as described in our previous study,[6] the multiplex assay was applied to differentiate multiple isolates from 22 patients (89 isolates) treated by azole therapy (fluconazole). Unchanged genotype was observed in 63.6% of cases (14 patients). Among them, only four patients (patients 11, 14, 17 and 19) under fluconazole therapy, developed resistance to the drug. Highly related multilocus genotype was detected in 13.6% of cases (patients 3, 7 and 20) with unchanged susceptibility profile. A microvariation and a strain replacement occurred simultaneously in patient 3. On the other hand, a strain replacement was observed in six patients (27.2%). Among them, two patients (patient’s n°3 and 10) showed selected different genotype type with reduced susceptibility to fluconazole (MIC≥64 µg/mL) [table I]. In the present study, we evaluated mechanisms of resistance to fluconazole cited above by comparing the gene expression profiles of CgCDR1, CgCDR2, and CgERG11 in susceptible C. glabrata isolates (n=10) versus resistant ones to fluconazole (n=6) referring to URA3 gene (tables II and III). We found that 14 from 16 susceptible isolates expressed basal levels which were below threshold value of 2.5 compared to levels of expression by control isolate TU10. Considering that the first isolate recovered before treatment was fluconazole susceptible and the second isolate obtained from the same patient after treatment was fluconazole resistant, we documented

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the apparition of fluconazole resistance in six patients (n° 3, 10, 11, 14, 17 and 19). As shown in table I, strains from patient’s n° 11, 14, 17 and 19 developed reduced fluconazole susceptibility during treatment while maintaining identical microsatellite genotype. For the four patients, the times that elapsed between the time of sampling of the azole-susceptible isolate and the time of sampling of the azoleresistant isolate were 150, 30, 220, and 120 days, respectively. At these times, the patients had received cumulative doses of 4, 5.6, 8 and 12 g of fluconazole, respectively. Susceptible and resistant isolates of the same type were analyzed in these patients. In patient n° 11, a 3-fold increase was observed in CgCDR2 expression relative to susceptible isolate. In patient n°19, an over-expression of CgCDR2 (85 fold) was reported in the resistant isolate (table III). Therefore, the pre-treatment susceptible isolate from patient 14 was not amplified by the qPCR, whereas resistant isolate showed high degree of expression in both CgCDR1 and CgCDR2 (table III). A selected resistant isolate (3.2) from patient 3 over-expressed all three genes CgCDR1, CgCDR2 and CgERG11 by 2.54, 375.57 and 4.34 fold, respectively. However, two resistant isolates 10.2 and 17.3 did not over-expressed these genes.

4. Discussion It should be emphasized that determination of pre- and posttreatment drug susceptibility of pathogens requires genotyping of the strains in order to distinguish between recolonization by a new strain and the development or selection of resistant strains. The degree of genetic variability detected between pre- and post-treatment C. glabrata isolates strongly depends on the molecular procedure used and on the individual patient. Microsatellite analysis of four polymorphic markers was the method of genotyping employed previously described by the authors and which provided high discriminatory power between related isolates.[6-9] Some isolates from unrelated patients presented identical genotypes and similar MIC to fluconazole (table II). Our sample collection of isolates was obtained from a restricted geographical area, which could partially explain the predominance of some genotypes, as described previously, or selective ecological advantages might exist.[19,20] Only six patients have resistant isolates after fluconazole therapy. Molecular typing revealed that pre- and post-treatment genotype was identical in four patients. Moreover, in two patients, resistant isolates were genotypically different from susceptible ones. Apparently, resistant strains were selected from a pool of colonizing strain or a recolonization process was done in patient’s n°3 and 10 where a strain with a lower resistance managed to be replaced by a more resistant strain. In many cases, distinction between recolonization and strain selection was difficult when a precise definition of the phenomenon of clonal replacement is generally missing from the literature. In the present communication, we considered strain selection a likely event as these two patients were under antifungal drugs.

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Table I. Genotypes of sequential isolates from Candida glabrata infections.

No. of Patient

No. of episodes

No. of isolates(a)

MIC FCZ (µg/mL)

Anatomical site

Microsatellite analysis (Size in bp)

FCZ treatment(b) (d0-dn)

MTI

ERG3

GLM4

GLM5

1

5

1

4

Urine

246

190

267

262

d12-d25

2

3

1 2

0.125 2

Urine

238 240

197 204

273 264

304 259

d1-d21

1 2 3

8 256 6

Urine

238 238 238

197 197 197

273 288 273

304 271 298

d13-d60

3

6

4

3

1

2

Urine

238

197

273

298

d35-d59

5

2

1

256

Vagina

240

204

264

259

d7-d10

6

4

1

4

Urine

246

190

267

262

d4-d14

7

9

1 2

6 0.016

Urine

238 238

197 197

288 276

271 271

d5-d25

8

2

1 2

256 3

Urine

238 238

197 197

288 288

271 271

d5-d9

9

7

1 2 3

4 2 1.5

Urine

238 238 238

197 197 197

267 276 273

262 259 298

d5-d15

10

5

1 2 3

1.5 32 256

Urine

238 240 238

197 197 197

273 264 288

298 259 271

d60-d120

11

3

1 2

1.5 256

Urine

238 238

197 197

273 273

298 298

d35-d55

12

2

1

4

Urine

240

205

264

259

d12-d22

13

2

1 2

0.125 2

Urine

240 238

200 228

273 273

298 298

d10-d17

14

2

1 2

16 256

Urine

227 227

227 227

276 276

262 262

d2-d30

15

7

1

4

Urine

240

204

264

259

d5-d12

16

2

1

1.25

Urine

238

197

273

298

d4-d11

17

6

1 2 3 4

6 8 256 0.25

Vagina

240 238 238 238

204 197 197 197

264 288 288 273

259 271 271 298

d45-d65 d120-d180

18

4

1 2

1 3

Vagina

238 240

198 204

273 273

298 307

d11-d18

19

2

1 2

4 256

Urine

238 238

197 197

270 270

271 271

d20-d40 d60-d100

20

3

1 2

1.5 2

Urine

238 238

228 197

273 273

298 298

d15-d30

21

5

1

2

Urine

239

227

276

262

d17-d22

22

5

1

1

Urine

238

197

273

298

d7-d17

a: isolates are presented in chronologic order; b: fluconazole treatment is given in days; d0: the first positive sample d: day; ERG3: C-5 sterol desaturase; FCZ: fluconazole; GLM4: glabrata microsatellite markers 4; GLM5: glabrata microsatellite markers 5; MIC FCZ: minimal inhibitory concentration to fluconazole; MTI: metallothionein I (MTI) gene.

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Table II. Expression profiles of resistance-related genes for the 10 fluconazole-sensitive C. glabrata unrelated isolates.

Microsatellite analysis (size in bp)

Gene expression (fold increase)

Isolatesa

Isolation site

MTI

ERG3

GLM4

GLM5

MIC (µg/mL)

CDR1RNA

CDR2RNA

ERG11RNA

1

Urine

239

260

276

259

0.125

0.04

0.79

0.38

2

Urine

240

203

264

259

4.000

0.82

0.96

1.32

3

Urine

240

203

264

259

4.000

1.81

2.79

0.52

4

Urine

240

205

264

259

4.000

0.12

0.29

0.43

5

Urine

238

197

273

301

0.500

0.03

1.61

0.09

6

Urine

238

197

273

298

8.000

0.01

0.57

1.89

7

Urine

238

197

273

301

1.000

0.43

0.46

0.09

8

Urine

238

197

273

301

2.000

0.30

0.26

0.16

9

Urine

238

197

273

298

4.000

1.59

1.13

0.74

10

Urine

240

203

264

259

4.000

0.86

0.74

0.71

a: isolates were collected from patients that have never exposed to fluconazole. CDR1RNA: Candida glabrata cerebellar degeneration-related protein 1 ribonucleic acid; CDR2RNA: Candida glabrata cerebellar degenerationrelated protein 2 ribonucleic acid; ERG3: C-5 sterol desaturase; ERG11RNA: sterol 14 alpha-demethylase Erg 11 ribonucleic acid; FCZ: fluconazole; GLM4: glabrata microsatellite markers 4; GLM5: glabrata microsatellite markers 5; MIC FCZ: minimal inhibitor concentration to fluconazole; MTI: metallothionein I (MTI) gene. Table III. Molecular typing and gene expression analyses for patients who developed resistance to fluconazole in the course of antifungal treatment.

No. of Patient

No. of isolats

Isolation site

Antifungaltreatment Isolation MIC FCZ (cumulative dose in day (j0-jn) (µg/mL) gramme)

Molecular typing by microsatellite markers (size in bp): MTI ERG3 GLM4 GLM5

RNA expression level of

CDR1

CDR2

ERG11

11

11-1 11-2

Urine

0 150

1.5 256

4

238 238

197 197

273 273

298 298

3.09 5.64

6.62 20.04

0.4 0.54

14

14-1 14-2

Urine

0 30

16 256

5.6

227 227

228 228

276 276

262 262

ND 92.61

ND 169.65

ND 0.12

17

17-2 17-3

Vagin

0 220

8 256

8

238 238

197 197

288 288

271 271

0.12 0.29

0.44 0.57

0.24 0.16

19

19-1 19-2

Urine

0 120

4 256

12

238 238

197 197

270 270

271 271

ND 0.1

0.11 9.42

0.53 0.3

3

3-1 3-2 3-3

Urine

0 60 80

8 256 8

9.6

238 238 238

197 197 197

273 288 273

304 271 298

0.1 2.54 0.03

0.49 375.57 0.67

0.94 4.34 1.85

10

10-1 10-2

Urine

0 150

1.5 256

12

238 238

197 197

273 288

298 271

0.01 0.31

0.57 1.2

1.89 1.67

CDR1: Candida glabrata cerebellar degeneration-related protein 1; CDR2: Candida glabrata cerebellar degeneration-related protein 2; ERG3: C-5 sterol desaturase; ERG11: sterol 14 alpha-demethylase; FCZ: fluconazole; GLM4: glabrata microsatellite markers 4; GLM5: glabrata microsatellite markers 5; MIC FCZ: minimal inhibitor concentration to fluconazole; MTI: metallothionein I (MTI) gene; ND: not documented; RNA: ribonucleic acid.

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Several authors reported that fluconazole antifungal therapy or prophylaxis was the main mechanism implicated in fluconazole resistance[21,22] as also shown in our study when 6 of 22 patients (27%) under fluconazole therapy acquired reduced susceptibility strains.[23] Despite the limited number of patients studied, fluconazole seems to play the role of monitor that leads to fluconazole resistance in our collection of isolates and the minimal fluconazole dose administered in these patients was 200 mg daily during one month (patient 14). The resistance development was associated with increased gene expression of CgCDR1 and CgCDR2 in patient n°11 and 14. The concomitant hyper-expression of CDR1 and CDR2 genes in some resistant isolates was not surprising because these two genes are regulated by the same transcription factor CgPDR1 (not studied in this work).[24-26] In resistant isolates from patient n° 17 (isolate n°17.3) and from patient n°10 (isolate n°10.2), no variation in gene expression was observed. Resistance of C. glabrata isolates could be related to the existence of other mechanisms such as mutations in the CgERG11 gene or another membrane transporter SNQ2 which were not studied in this work. Only over-expression of CgERG11 gene was found in a resistant isolate of C. glabrata[11] and, Marichal et al. showed that gene duplication is responsible for the overproduction.[27] Norbert et al. described different mutations in CgERG11 in five C.glabrata isolates but heterologous expression of the CgERG11 mutant allele did not provide evidence for its involvement in azole resistance.[28] Somes isolates (n°3 table II and 11.1 table III) showed over-expression of CgCDR2 and/or CDR1 gene. This can be either an experimental artefact or a response to other environmental factors. In conclusion, the present study demonstrated that C. glabrata populations inhabiting in vivo are subject to frequent changes. The causative selection and/or acquisition events may be clinically relevant processes since they reflect the capacity of a C. glabrata strain to respond to environmental signals to develop fluconazole resistance. Our study confirms also the role of upregulation of the ABC efflux transporters CgCDR1 and CgCDR2 as a major mechanism of fluconazole resistance in C. glabrata. Moreover, other genes implicated can be explored in future studies.

Acknowledgements This study was financially supported by the Minister of High Education and Scientific Research and Aix Marseille University. No conflict of interest and approval state was not required.

cerebellar degeneration-related protein 2; CLSI: clinical and laboratory standars institute; DNA: deoxyribonucleic acid; ERG3: C-5 sterol desaturase; ERG11: sterol 14 alpha-demethylase Erg11; FCZ: fluconazole; MIC: minimal inhibitrice concentration; PCR: polymerase chain reactions; q-PCR: real time PCR; RNA: ribonucleotidic acid; RT-PCR: reverse transcriptase-PCR; TU10: reference strain with low susceptibility to fluconazole; URA3: urotidine 5-phosphate decarboxylase; YEPD: yeast extract peptone dextrose.

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Funding. No specific funding was received for this study.

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Conflicts of interests. None.

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Abbreviations. Bp: base pairs; CgCDR1: Candida glabrata cerebellar degeneration-related protein 1; CgCDR2: Candida glabrata

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Candida Glabrata Fluconazole Resistance

16. Wada S, Niimi M, Niimi K, et al. Candida glabrata ATP-binding cassette transporters Cdr1p and Pdh1p expressed in a Saccharomyces cerevisiae strain deficient in membrane transporters show phosphorylation-dependent pumping properties. J Biol Chem 2002; 277: 46809-21 17. Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard CLSI document M27-A3. 2008 18. Peirson SN, Butler JN, Foster RG. Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids Res 2003; 31: e73 19. Dodgson AR, Pujol C, Denning DW, et al. Multilocus sequence typing of Candida glabrata reveals geographically enriched clades. J Clin Microbiol 2003; 41 : 5709-17 20. Abbes S, Sellami H, Sellami A, et al. Microsatellite analysis and susceptibility to fluconazole of Candida glabrata invasive isolates in Sfax Hospital, Tunisia. Medical Mycology 2011; 49: 10-5 21. Lin CY, Chen YC, Lo HJ, et al. Assessment of Candida glabrata strain relatedness by pulsed field gel electrophoresis and multilocus sequence typing. J Clin Microbiol 2007; 45: 2452-9 22. Tumbarello M, Posteraro B, Trecarichi EM, et al. Fluconazole use as an important risk factor in the emergence of fluconazole-resistant Candida glabrata fungemia. Arch Intern Med 2009; 169: 1444-5

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23. Abbes S, Sellami H, Sellami A, et al. Bases de la résistance au fluconazole des souches de Candida glabrata isolées au CHU de Sfax, Tunisie. J Myc Med 2012; 118 doi: 10.1016/j.mycmed.2011.12.060 24. Ferrari S, Ischer F, Calabrese D, et al. Gain of function mutations in CgPDR1 of Candida glabrata not only mediate antifungal resistance but also enhance virulence. PLoS Pathog 2009; 5: e1000268 25. Paul S, Schmidt JA, Moye-Rowley WS. Regulation of the CgPdr1 transcription factor from the pathogen Candida glabrata. Eukaryotic Cell 2011; 10: 187-97 26. Torelli R, Posteraro B, Ferrari S, et al. The ATP-binding cassette transporterencoding gene CgSNQ2 is contributing to the CgPDR1-dependent azole resistance of Candida glabrata. Mol Microbiol 2008; 68: 186-201 27. Marichal P, Vanden Bossche H, Odds FC, et al. Molecular biological characterization of an azole-resistant Candida glabrata isolate. Antimicrob Agents Chemother 1997; 41: 2229-37 28. Norbert B, Borecka S, Dzugasova V, et al. Mutations in the CgPDR1 and CgERG11 genes in azole-resistant Candida glabrata clinical isolates from Slovakia. Int J Antimicrob Agents 2009; 33: 574-8 Correspondence and offprints: Ayadi Ali, Laboratoire de biologie moléculaire, parasitaire et fongique, Faculté de médecine, 3029 Sfax, Tunisie. E-mail: [email protected]

Thérapie 2014 Septembre-Octobre; 69 (5)