Objectives: Several studies have documented the potent in vitro activity of caspofungin against Candida spp. This is of special concern for Candida glabrata infections that are often resistant to many azole antifungal agents and, consequently, difficult to treat. The aim of the present study was to expand the data on the in vitro activity of caspofungin against azole-resistant isolates of C. glabrata. Methods: A total of 50 clinical isolates of C. glabrata were tested for susceptibility to caspofungin. The isolates were cross-resistant to multiple azoles, including fluconazole, itraconazole, ketoconazole and voriconazole. Expression of the resistance-related CgCDR1 and CgCDR2 genes was evaluated by quantitative RT-PCR analysis. The MICs of caspofungin were determined by using the National Committee for Clinical Laboratory Standards M27-A2 reference method. Results: C. glabrata isolates exhibited increased expression of the CDR efflux pump(s), and this was in accordance with their high-level azole resistance. In contrast, all the isolates were highly susceptible to caspofungin (100% of isolates were inhibited at ≤1 mg/L). Conclusions: Our results represent further evidence for the excellent antifungal potency of caspofungin, particularly against C. glabrata isolates expressing cross-resistance to azole

Fadda, Giovanni

Fiori, Barbara

La Sorda, Marilena

Posteraro, Brunella

Sanglard, Dominique

Sanguinetti, Maurizio

Spanu, Teresa

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Journal of Antimicrobial Chemotherapy (2006) 58, 458–461doi:10.1093/jac/dkl237Advance Access publication 6 June 2006Caspofungin activity against clinical isolates of azole cross-resistantCandida glabrata overexpressing efflux pump genesBrunella Posteraro1, Maurizio Sanguinetti1*, Barbara Fiori1, Marilena La Sorda1, Teresa Spanu1,Dominique Sanglard2 and Giovanni Fadda11Istituto di Microbiologia, Universita` Cattolica del Sacro Cuore, Rome, Italy; 2Institut de Microbiologie,Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, SwitzerlandReceived 16 March 2006; returned 14 April 2006; revised 10 May 2006; accepted 12 May 2006Objectives: Several studies have documented the potent in vitro activity of caspofungin against Candidaspp. This is of special concern for Candida glabrata infections that are often resistant to many azoleantifungal agents and, consequently, difficult to treat. The aim of the present study was to expand thedata on the in vitro activity of caspofungin against azole-resistant isolates of C. glabrata.Methods: A total of 50 clinical isolates of C. glabrata were tested for susceptibility to caspofungin. Theisolates were cross-resistant to multiple azoles, including fluconazole, itraconazole, ketoconazole andvoriconazole. Expression of the resistance-related CgCDR1 and CgCDR2 genes was evaluated byquantitativeRT–PCR analysis. TheMICs of caspofunginwere determined by using theNational Committeefor Clinical Laboratory Standards M27-A2 reference method.Results: C. glabrata isolates exhibited increased expression of the CDR efflux pump(s), and this was inaccordance with their high-level azole resistance. In contrast, all the isolates were highly susceptible tocaspofungin (100% of isolates were inhibited at £1 mg/L).Conclusions: Our results represent further evidence for the excellent antifungal potency of caspofungin,particularly against C. glabrata isolates expressing cross-resistance to azoles.Keywords: antifungals, susceptibility testing, CgCDR genesIntroductionCandida glabrata infections have markedly increased in the pastdecades and, when invasive, are associated with a high mortalityrate, especially in immunocompromised patients such asintensive-care, post-surgical and neutropenic patients.1 This, inpart, has been attributed to the reduced susceptibility of thispathogen to antifungal agents, especially the azoles.2 Recentstudies revealed that the MICs of extended-spectrum triazoles(fluconazole, itraconazole and voriconazole) for C. glabratawere higher than those seen for most Candida albicans isol-ates.3–6 Fluconazole-resistant isolates have been found not onlyin AIDS patients with oropharyngeal candidiasis, but also infungaemic patients and among vaginal isolates as a consequenceof widespread and often indiscriminate use of the drug.7 C. glab-rata is able to rapidly develop azole resistance, which is probablyrelated to the haploid state of the microorganism.2 In this context,studying the resistance mechanisms of this pathogenic yeast mayprovide answers in developing new antifungal agents.6,8Caspofungin is an echinocandin with potent fungicidal activityagainst many Candida species3,9 and could be particularly usefulto treat C. glabrata infections, which often develop resistance toazoles. To further demonstrate the in vitro efficacy of thispromising new antifungal agent in C. glabrata, we report herecaspofungin susceptibility of a large collection of clinical isolateswhich were cross-resistant to multiple azoles. Molecular studiesshowed that azole resistance in these isolates is associated withthe up-regulation of the ABC transporter genes CgCDR1 andCgCDR2.Materials and methodsYeast isolates and antifungal susceptibility testingA total of 50 clinical isolates of azole-resistant C. glabrata weretested: 30 isolates were recovered from blood (4 isolates), respiratorytract (10), vagina (3), mouth (1), urine (10), skin (1) and drainagefluid (1) of patients hospitalized between January 2004 and June.............................................................................................................................................................................................................................................................................................................................................................................................................................*Corresponding author. Tel: +39-06-30154964; Fax: +39-06-3051152; E-mail: msanguinetti@rm.unicatt.it.............................................................................................................................................................................................................................................................................................................................................................................................................................458 The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.For Permissions, please e-mail: journals.permissions@oxfordjournals.org2005, and 20 other isolates, cultured from various patient specimens(6 blood, 5 urine, 3 vagina, 2 respiratory tract, 2 mouth, 1 skin and 1drainage fluid) before 2004, had been explored for molecular mech-anisms of antifungal resistance in a previous study.6 Each isolaterepresented an individual infectious episode. The isolates showedhigh MICs of multiple azoles, as determined by the broth microdi-lution method (Table 1). A panel of 30 fluconazole-susceptible orfluconazole-susceptible dose-dependent (S-DD) isolates was alsoincluded. All isolates were identified to the species level by standardmethods10 and stored as 20% glycerol stocks until use. Prior totesting, each isolate was subcultured onto Sabouraud dextroseagar. C. glabrata ATCC 36909 reference strain and two well-char-acterized C. glabrata isolates, DSY562 (a susceptible isolate) andDSY565 (a resistant isolate),11 were included as controls. Antifungalsusceptibility testing was performed by the broth microdilutionmethod in RPMI with 2% glucose according to the M27-A2 docu-ment of the Clinical and Laboratory Standards Institute (CLSI, for-merly the NCCLS).12 The inoculum suspensions were adjustedspectrophotometrically at 530 nm to match the concentration of1.5 – 1.0 · 103 cells/mL. Stock solutions of caspofungin (Merck& Co., Whitehouse Station, PA, USA), fluconazole (Pfizer, Inc.,New York, NJ, USA), itraconazole (Janssen, Beerse, Belgium), keto-conazole (Sigma Aldrich, Milan, Italy) and voriconazole (Pfizer)were prepared in water (caspofungin and fluconazole) or dimethylsulphoxide (itraconazole, ketoconazole and voriconazole). The finalconcentrations of the antifungal agents were 0.008–8 mg/Lfor caspofungin, itraconazole, ketoconazole and voriconazole and0.125–128 mg/L for fluconazole. The trays were incubated at35C, and MIC end points were read at 48 h. All tests were carriedout in duplicate. For caspofungin, the MIC end point was defined asthe lowest concentration determining complete inhibition of growth.This end point was chosen based on the fungicidal activity of caspo-fungin as already described.9 For azoles, the MIC end point wasdefined as the lowest concentration that produced a prominentdecrease in turbidity (80% inhibition) compared with that of thegrowth control. The interpretive criteria used for fluconazole, itra-conazole and voriconazole were those published by the CLSI.12,13Interpretive breakpoints have not yet been established for eitherketoconazole or voriconazole, but we used susceptibility breakpointsof £0.125 and £1 mg/L, respectively.6 The CLSI quality controlstrains Candida krusei ATCC 6258 and Candida parapsilosisATCC 22019 were tested and MICs were within the expected range.Rhodamine 6G accumulation assayThe rhodamine 6G accumulation assay was performed as describedpreviously.11,14 Following growth overnight in YEPD (1% yeastextract, 2% peptone, 2% glucose) liquid medium at 30C, yeastcells were diluted 100-fold in 5 mL of the same medium. The culturewas grown to reach a cell density of 107 cells/mL, then placed at4C and divided into 1 mL aliquots. Labelling of the cells (107) with10 mM rhodamine 6G (Societa` Italiana Chimici, Rome, Italy) wasperformed under constant shaking for 30 min at 30C, then stoppedby placing reaction tubes on ice. The labelled cells were diluted 40-fold in ice-cold 0.1 M phosphate-buffered saline (PBS) at pH 7.0 andthen subjected to flow cytometry using a Coulter EPICS XL instru-ment (Beckman Coulter, Brea, CA, USA). Experiments wererepeated two times.Expression levels of ABC transporter genesQuantitative expression of the CgCDR1 and CgCDR2 genes wasperformed by real-time RT–PCR analysis with an i-Cycler iQ system(Bio-Rad Laboratories, Hercules, CA, USA), following a previouslydescribed protocol.6 Relative quantification of the target gene wascarried out by using a primer pair and a Taqman probe in combina-tion with primers and a probe specific for the URA3 reference gene.Each reaction was performed in triplicate. For each isolate, foldincreases in gene expression were calculated from the meannormalized expression relative to the mean normalized expressionof the DSY562 isolate, used as a susceptible control.15 Genes with aDCT value, calculated as CT [test gene]–CT [URA3], that fell overthe 3-SD range measured in the azole-susceptible isolates wereconsidered overexpressed, as reported previously.16Results and discussionTable 1 summarizes the in vitro susceptibility of 80 C. glabrataclinical isolates to caspofungin, itraconazole, ketoconazole andvoriconazole stratified by fluconazole susceptibility category.With respect to fluconazole MIC, the isolates fell into the fol-lowing groups: susceptible (15 isolates), S-DD (15 isolates) andresistant (50 isolates). All fluconazole-resistant isolates were alsocross-resistant to the other azoles tested (Table 1), and thisphenotype was found to be associated with the overexpressionof drug efflux pump-encoding CgCDR genes. The expression ofCgCDR1 and CgCDR2 was strongly increased in the majority ofthe 50 azole-resistant isolates compared with that of the suscept-ible control isolate DSY562, but not in the 15 azole-susceptibleisolates studied (Figure 1). Of the resistant isolates 46 exhibited12.4- to 483.0-fold up-regulation of the ABC transporter geneCgCDR1. Notably, 27 of these 46 also exhibited 3.9- to 70.6-foldup-regulation of CgCDR2, with the remaining 4 isolates showing26.1- to 61.2-fold up-regulation of this second ABC transportergene. The 15 fluconazole-S-DD isolates up-regulated CgCDR1 orCgCDR2 (Figure 1), but the levels of up-regulation were signi-ficantly less than those by the resistant isolates studied (109.9- to5.5-fold for CgCDR1 and 17.4- to 2.6-fold for CgCDR2).Furthermore, the increased expression of efflux pumps in thefluconazole-resistant and fluconazole-S-DD isolates was alsoaccompanied by decreased accumulation of rhodamine 6G, asshown by their mean fluorescence values, which differed fromTable 1. In vitro activity of caspofungin and azole antifungal agentsagainst 80 clinical isolates of C. glabrata stratified by fluconazolesusceptibilityFluconazolesusceptibilitycategoryaNo.testedAntifungalagentMIC range(mg/L)MIC50(mg/L)MIC90(mg/L)S 15 caspofungin 0.03–0.125 0.06 0.125itraconazole 0.125–0.5 0.25 0.5ketoconazole 0.06–0.25 0.125 0.25voriconazole 0.06–0.25 0.125 0.125S-DD 15 caspofungin 0.03–0.25 0.125 0.25itraconazole 0.125–2 0.5 1ketoconazole 0.06–0.5 0.125 0.5voriconazole 0.125–1 0.25 0.5R 50 caspofungin 0.06–1 0.25 1itraconazole 2–>8 8 >8ketoconazole 1–8 4 4voriconazole 2–8 4 8aS, MIC £ 8 mg/L; S-DD, MIC = 16–32 mg/L; R, MIC ‡ 64 mg/L.Caspofungin against azole-resistant Candida glabrata459those of the susceptible isolates by 96.1- and 37.0-fold,respectively (Figure 1). In contrast, caspofungin was very activein vitro against all the fluconazole-resistant and fluconazole-S-DD isolates and, as expected, against the fluconazole-susceptibleisolates. MIC90 (MIC at which 90% of the isolates wereinhibited) of fluconazole-resistant isolates was 1 mg/L. MIC90sof fluconazole-S-DD isolates and fluconazole-susceptible isolateswere 0.25 and 0.125 mg/L, respectively. For all the isolates, theMIC of caspofungin was £1 mg/L. Low MICs (0.06 mg/L) ofcaspofungin were also obtained for the C. glabrata referencestrain, ATCC 36909, and two well-characterized C. glabrataisolates, DSY562 and DSY565.Cross-resistance within a class of antimicrobial agents is animportant issue, especially if therapeutic options are limited by apoor number of available compounds as for the antifungal agents.Although the new systemic antifungal triazoles (ravuconazole andvoriconazole) are more active in vitro than fluconazole and itra-conazole against Candida spp.,17 cross-resistance between fluc-onazole and ravuconazole has been observed, so that fluconazolewas recently proposed as a surrogate marker to predict thesusceptibility of Candida spp. to ravuconazole.18 The cross-resistance involving ravuconazole,18 like that involvingvoriconazole,4,6 applies most directly to fluconazole-resistantC. glabrata. As stated by Sobel,7 C. glabrata can be consideredthe Achilles heel of all available azoles, thus C. glabratainfections treated with these drugs are difficult to eradicate.Our results showed that caspofungin had excellent activityagainst fluconazole (multiazole)-resistant isolates, as well asagainst isolates susceptible and dose-dependent susceptible tofluconazole. This is not surprising considering that the echino-candin caspofungin belongs to a class of antifungal agents that actby inhibiting the cell wall glucan synthesis. Interestingly,fluconazole-resistant isolates showed a higher MIC90 valuecompared with fluconazole-S-DD or fluconazole-susceptibleisolates. This difference indicates that azole resistance cancontribute to an increase in caspofungin MIC. The basis forsuch a difference is still unknown.In conclusion, the present study confirms the efficacy ofcaspofungin against clinical isolates of C. glabrata, but, forthe first time, the potent activity of this drug was assessedwith a large number of C. glabrata isolates whose cross-azoleresistance was well documented by molecular methods. Further-more, these findings support the use of caspofungin to treat thoseC. glabrata infections for which amphotericin B represents thesole therapeutic choice.AcknowledgementsThis work was supported by grants from the Istituto Superiore diSanita` (National Program of Research on AIDS, No. 50 F.31) andfrom the Universita` Cattolica del S. Cuore (Fondi Ateneo LineaD1-2005).Transparency declarationsNone to declare.References1. Marr KA. Invasive Candida infections: the changing epidemiology.Oncology 2004; 18: 9–14.100010010Fold increase10.1R S-DD SR S-DD SR S-DD S100101Fold increase0.1100101Mean fluorescence0.1(a)(b)(c)Figure 1. Expression of ABC transporters CgCDR1 (a) and CgCDR2 (b) andrhodamine 6G accumulation (c) in C. glabrata clinical isolates. With respect tofluconazole, isolates were defined as resistant (R), susceptible-dose dependent(S-DD) or susceptible (S). Fold increases in gene expressionwere determined byRT–PCR analysis relative to DSY562. Fluorescence values were determined byflow cytometry. The mean value (a and b) or the geometric mean value (c) foreach group is indicated by a dark solid horizontal line.Posteraro et al.4602. Fidel PL, Vazquez JA, Sobel JD. Candida glabrata: review ofepidemiology, pathogenesis, and clinical disease with comparison toC. albicans. Clin Microbiol Rev 1999; 12: 80–96.3. Pfaller MA, Diekema DJ, Messer SA et al. In vitro activities ofcaspofungin compared with those of fluconazole and itraconazoleagainst 3,959 clinical isolates of Candida spp., including 157fluconazole-resistant isolates. Antimicrob Agents Chemother 2003; 47:1068–71.4. Pfaller MA, Messer SA, Boyken L et al. Geographic variation in thesusceptibilities of invasive isolates of Candida glabrata to sevensystemically active antifungal agents: a global assessment from theARTEMIS Antifungal Surveillance Program conducted in 2001 and2002. J Clin Microbiol 2004; 42: 3142–6.5. Hajjeh RA, Sofair AN, Harrison LH et al. Incidence of bloodstreaminfections due to Candida species and in vitro susceptibilities of isolatescollected from 1998 to 2000 in a population-based active surveillanceprogram. J Clin Microbiol 2004; 42: 1519–27.6. Sanguinetti M, Posteraro B, Fiori B et al. Mechanisms of azoleresistance in clinical isolates of Candida glabrata collected during ahospital survey of antifungal resistance. Antimicrob Agents Chemother2005; 49: 668–79.7. Sobel JD. Management of infections caused by Candida glabrata.Curr Infect Dis Rep 2000; 2: 424–8.8. Borst A, Raimer MT, Warnock DW et al. Rapid acquisition ofstable azole resistance by Candida glabrata isolates obtained before theclinical introduction of fluconazole. Antimicrob Agents Chemother 2005;49: 783–7.9. Pfaller MA, Messer SA, Boyken L et al. Caspofungin activityagainst clinical isolates of fluconazole-resistant Candida. J Clin Microbiol2003; 41: 5729–31.10. Warren NG, Hazen KC. Candida, Cryptococcus, and otheryeasts of medical importance. In: Murray PR, Baron EJ, Pfaller MA,Tenover FC, Yolken RH, eds. Manual of Clinical Microbiology, 7th edn.Washington, DC, USA: ASM Press; 1184–99.11. Sanglard D, Ischer F, Calabrese D et al. The ATP binding cassettetransporter gene CgCDR1 from Candida glabrata is involved in theresistance of clinical isolates to azole antifungal agents. AntimicrobAgents Chemother 1999; 43: 2753–65.12. National Committee for Clinical Laboratory Standards. ReferenceMethod for Broth Dilution Antifungal Susceptibility Testing of Yeasts:Approved Standard M27-A2. NCCLS, Wayne, PA, USA, 2002.13. Pfaller MA, Diekema DJ, Rex JH et al. Correlation of MIC withoutcome for Candida species tested against voriconazole: analysis andproposal for interpretive breakpoints. J Clin Microbiol 2006; 44: 819–26.14. Posteraro B, Sanguinetti M, Sanglard D et al. Identification andcharacterization of a Cryptococcus neoformans ATP binding cassette(ABC) transporter-encoding gene, CnAFR1, involved in the resistance tofluconazole. Mol Microbiol 2003; 47: 357–71.15. Peirson SN, Butler JN, Foster RG. Experimental validation ofnovel and conventional approaches to quantitative real-time PCR dataanalysis. Nucleic Acids Res 2003; 31: e73.16. Chau AS, Mendrick CA, Sabatelli FJ et al. Application of real-time quantitative PCR to molecular analysis of Candida albicans strainsexhibiting reduced susceptibility to azoles. Antimicrob AgentsChemother 2004; 48: 2124–31.17. Pfaller MA, Messer SA, Hollis RJ et al. In vitro activities ofravuconazole and voriconazole compared with those of four approvedsystemic antifungal agents against 6,970 clinical isolates of Candidaspp. Antimicrob Agents Chemother 2002; 46: 1723–7.18. Pfaller MA, Messer SA, Boyken L et al. Cross-resistance betweenfluconazole and ravuconazole and the use of fluconazole as a surrogatemarker to predict susceptibility and resistance to ravuconazole among12,796 clinical isolates of Candida spp. J Clin Microbiol 2004; 42:3137–41.Caspofungin against azole-resistant Candida glabrata461

Caspofungin activity against clinical isolates of azole cross-resistant Candida glabrata overexpressing efflux pump genes

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