The increasing use of azole antifungals for the treatment of mucosal and systemic Candida glabrata i.docx

1、The increasing use of azole antifungals for the treatment of mucosal and systemic Candida glabrata iMechanisms of Azole Resistance in Clinical Isolates of Candidaglabrata Collected during a Hospital Survey ofAntifungal ResistanceThe increasing use of azole antifungals for the treatment of mucosal an

2、d systemic Candida glabrata infections has resulted in the selection and/or emergence of resistant strains. The main mechanisms of azole resistance include alterations in the C. glabrata ERG11 gene (CgERG11), which encodes the azole target enzyme, and upregulation of the CgCDR1 and CgCDR2 genes, whi

3、ch encode efflux pumps. In the present study, we evaluated these molecular mechanisms in 29 unmatched clinical isolates of C. glabrata, of which 20 isolates were resistant and 9 were susceptible dose dependent (S-DD) to fluconazole. These isolates were recovered from separate patients during a 3-yea

4、r hospital survey for antifungal resistance. Four of the 20 fluconazole-resistant isolates were analyzed together with matched susceptible isolates previously taken from the same patients. Twenty other azole-susceptible clinical C. glabrata isolates were included as controls. MIC data for all the fl

5、uconazoleresistant isolates revealed extensive cross-resistance to the other azoles tested, i.e., itraconazole, ketoconazole, and voriconazole. Quantitative real-time PCR analyses showed that CgCDR1 and CgCDR2, alone or in combination, were upregulated at high levels in all but two fluconazole-resis

6、tant isolates and, to a lesser extent, in the fluconazole-S-DD isolates. In addition, slight increases in the relative level of expression of CgSNQ2 (which encodes an ATP-binding cassette ABC transporter and which has not yet been shown to be associated with azole resistance) were seen in some of th

7、e 29 isolates studied. Interestingly, the two fluconazole-resistant isolates expressing normal levels of CgCDR1 and CgCDR2 exhibited increased levels of expression of CgSNQ2. Conversely, sequencing of CgERG11 and analysis of its expression showed no mutation or upregulation in any C. glabrata isolat

8、e, suggesting that CgERG11 is not involved in azole resistance. When the isolates were grown in the presence of fluconazole, the profiles of expression of all genes, including CgERG11, were not changed or were only minimally changed in the resistant isolates, whereas marked increases in the levels o

9、f gene expression, particularly for CgCDR1 and CgCDR2, were observed in either the fluconazole-susceptible or the fluconazole-S-DD isolates. Finally, known ABC transporter inhibitors, such as FK506, were able to reverse the azole resistance of all the isolates. Together, these results provide eviden

10、ce that the upregulatlon of the CgCDR1-, CgCDR2-, and CgSNQ2-encoded efflux pumps might explain the azole resistance in our set of isolates. Candida glabrata has recently emerged as a significant pathogen in various hospital settings, where it is responsible for an increasing number of systemic infe

11、ctions and candiduria (2, 16). In a recent study, C. glabrata was the second most common non-C. albicans species as a cause of fungemia in the United States and was found to account for 21% of all Candida bloodstream isolates (26). Second only to C. albicans, C. glabrata is also the Candida species

12、most commonly recovered from the oral cavities of human immunodeficiency virus-infected patients (13, 16, 40).The rise in the number of C. glabrata systemic infections deserves a great deal of concern due to the high mortality rate associated with C. glabrata fungemia and to the propensity of this m

13、icroorganism to rapidly develop resistance to azole antifungal agents (10, 19). Several studies have revealed that a significant percentage of C. glabrata clinical isolates are resistant to fluconazole (approximately 9%) and itraconazole (37 to 40%) (3, 16, 25). More recently, in a surveillance stud

14、y conduced by pfaller et al. (27) to examine the antifungal susceptibilities of Candida species isolated from patients with bloodstream infections stratified by patient age, a trend of decreasing susceptibilities to fluconazole and itraconazole with increasing patient age was observed. In fact, none

15、 of the C. glabrata isolates from individuals :51 year old were resistant to fluconazole, whereas a higher proportion (5 to 9%) of resistant isolates was found in adult patients. Similarly, among 347 bloodstream, invasive, and colonizing strains Of C. glabrata isolated from patients at three urban t

16、eaching hospitals in New York City, the overall rates of resistance to fluconazole and itraconazole were 10.7 and 15.2%, respectively (33). The mechanisms of resistance to azole antifungal agents have been well elucidated in C. albicans and can be mainly categorized as (i) changes in the cell wall o

17、r plasma membrane, which lead to impaired azole uptake; (ii) alterations in the affinity of the drug target Ergl 1p (lanosterol 14a-demethylase) to azoles or in the cellular content of Ergl 1p due to target site mutation or overexpression of the ERG11 gene; and (iii) the efflux of drugs mediated by

18、membrane transport proteins belonging to the ATP-binding cassette (ABC) transporter family (CDR1 and CDR2) or to the major facilitator superfamily RI and FLU1). In the last case, the CDR1 and CDR2 sand the MDR1 gene were shown to be overexpressed in s resistant isolates, and deletion of these genes

19、resulted in insensitivity to azoles (34). In addition, compensatory path-that involve alterations of specific steps in ergosterol rithesis have been documented as mechanisms of resise to the azole and polyene antifungal classes (39).iore recently, increased levels of expression of the ABC /Toner gen

20、es C. glabrata CDR1 (CgCDR1) and CgCDR2 it been also shown in azole-resistant isolates of C. glabrata 15,35,36). Similar to C. albicans, genetic evidence support-* role of multidrug transporters in the azole resistance of ?brata was provided (36). Moreover, Marichal et al. (14) eusly showed increase

21、d levels of expression of ERG11 in vole-resistant C. glabrata strain which arose from a chronal duplication. In contrast, it has yet to be well explored er point mutations in the ERG11 gene are also impli- 0 in the resistance of C. glabrata to azoles.6e purpose of the present study was to determine

22、if the )cular mechanisms described above, alone or in combinawere sufficient to explain the phenotype of azole resisin unmatched clinical C. glabrata isolates obtained from pus clinical specimens during a 3-year hospital survey of tingal resistance or if other (not well-established) mechamight corre

23、late with azole resistance. In addition, pairs ;saceptible and resistant C. glabrata isolates that had been tined from the same patient and that had the same genotwere also examined. MATERIALS AND METHODS!isolates and growth conditions. The isolates of C. glabrata included in mot study were from a c

24、ollection of clinical isolates recovered during an .miologieal survey of antifungal resistance conducted at -ourinstitution, a university hospital in Rome, over a 3-year period (January 2000 through ber 2003). They were identified by standard methods (43) and tested for/7ceptibilities to amphoteriei

25、n B, flucytosine, fluconazole, ketoconazole, 1,.azole, and voriconazole by the Sensititre Yeast One commercial method urlical Service, Milan, Italy), as recommended by the manufacturer. Twena isolates for which fluconazole MICs exceeded the established susceptiilireakpoint (MIC 5 S pg/mt) (17, 32) w

26、ere retested for their antifungal nbilities by the NCCLS reference method for confirmation and were then d for molecular studies (see below). These isolates were recovered from body sites of 29 separate patients (Table 1). Among the isolates studied,(were 4 fluconazole-susceptible isolates that matc

27、hed 4 fluconazole-resisElates obtained from the same patient, as well as 20 randomly selected rtfl isolates of C. glabrata susceptible to azoles (Table 2). In addition, two idsracterized C. glabrata isolates, a susceptible isolate (isolate DSY562136j) rresistant isolate (isolate DSY565 1361), kindly

28、 furnished by Dominique bird, were used as controls. All 53 isolates were kept at -80C as 20% pal stocks and were subcultured, as required, on YEPD (1% yeast extract, splone, 2% glucose) agar plates at 30C. For liquid culture, the isolates gown in YEPD broth at 30C under constant agitation (240 rpm)

29、.dolgal susceptibility testing by the NCCLS reference method. Reference iogal susceptibility testing of the study isolates was performed by the broth Iliilution method described in NCCLS document M27-A2 (17). Powders of !eiikingal agents amphotericin B, flucytosine, ketoconazole. fluconazole, !anzol

30、e, and itraconazole were obtained from their respective manufacturHy, MICs were determined with RPMI 1640 with 2% glucose by use of Mitun size of 1.5 (1.0) x 103 cells/nil and incubation at 35C for 48 h. S control was ensured by testing the NCCLS-recommended strains C. ATCC 6258 and C. parapsilosis

31、ATCC 22019 (4). All tests were carried out licate. The interpretive criteria tot susceptibility to fluconazole, itraconund flucytosine were those published by Rex et al. (32) and the NCCLS 4dwere as follows: (i) for fluconazole, susceptible, 58 pg/ml; susceptible- !Ipendent (S-DD), 16 to 32 pg/m1; a

32、nd resistant, 64 g/m1; (ii) for zuole, susceptible, 50.125 pg/m1; S-DD, 0.25 to 0.5 pg/ml; and resistant, Quantitative real-time. RT-PCR. For quantitative real-time reverse transcription (RT)-PCR analysis, total RNA was extracted from C. glabrata cultures grown to the mid-exponential phase (optical

33、density at two nm OD,), approximately 0.6) with an RNAeasy Protect mini kit (Qiagen, Hilden, Germany), according to the instructions of the manufacturer, by mechanical disruption of the cells with glass beads and an RNase-free DNase treatment step. RNA integrity was assessed by determination of the OD.26/OD-, absorption ratio, and th