SUMMARY
We report a case of fluconazole-resistant oropharyngeal colonization caused by a strain of Candida glabrata that rapidly regained susceptibility once prophylaxis with this agent was discontinued and echinocandin therapy was initiated. Isolates collected before and after discontinuation of fluconazole were confirmed to be isogenic by RAPD analysis. Transcription analysis demonstrated constitutive expression of genes encoding efflux pumps in the isolate recovered on fluconazole prophylaxis and transient expression in those isolates collected after fluconazole was discontinued.
INTRODUCTION
Fluconazole prophylaxis is used routinely for prophylaxis during hemopoietic stem cell transplantation (HSCT). Emergence of fluconazole resistant Candida glabrata is increasing, necessitating the use of alternative antifungal therapy [1,2]. The development of resistance generally occurs following chronic exposure to an antifungal agent. Here, we report the rapid loss of fluconazole resistance in isogenic C. glabrata isolates from a HSCT patient following discontinuation of this azole followed by short-term echinocandin use.
A 58 year old male diagnosed with stage III IgG Kappa multiple myeloma was referred to the South Texas Veterans Health Care System (San Antonio, TX) Bone Marrow Transplant Program for an autologous HSCT. He had previously received localized radiation therapy to the spine and three cycles of thalidomide/dexamethasone and one cycle of salvage chemotherapy with bortezomib. The patient had received five months of fluconazole 200 mg/day since he was taking dexamethasone as part of his therapy. On admission, the fluconazole dose was increased to 400 mg/day. As part of the University of Texas Health Science Center at San Antonio Institutional Review Board approved research protocol, weekly oral fungal surveillance samples were collected, consisting of a 15 second swish with 10 mL of sterile water. Samples were plated on CHROMagar Candida plates for identification of fungal isolates and resistance screening [3, 4]. On the day of transplantation, the patient was switched to 50 mg of caspofungin due to an oral surveillance culture collected 3 days prior to transplantation that indicated colonization with fluconazole resistant C. glabrata (isolate 8127, MIC 64 µg/mL) as verified by CLSI M27-A3 methodology, performed in triplicate [5]. Four days later the patient was switched to Micafungin, dosed 100 mg daily for a 9 day course of prophylaxis. Ten days after transplantation, engraftment was reached and antifungal prophylaxis was discontinued. Interestingly, C. glabrata isolates 8140 and 8152 collected 6 and 14 days, respectively, after transplantation and after discontinuation of fluconazole were found to be fluconazole susceptible (MICs of 2 and 8 µg/mL, respectively) using CLSI M27-A3 methodology.
MATERIALS AND METHODS
To determine if these C. glabrata strains were isogenic, random amplification of polymorphic DNA (RAPD) analysis using previously described primers (OPA-18, OPE-18, and AP50-1) was utilized [6,7]. RAPD profiles were compared following PCR amplification with each other and C. glabrata ATCC 2001, an unrelated reference strain, using genomic DNA extracted from using the Masterpure Yeast DNA Purification Kit (Epicentre Biotechnologies, Madison, WI). Band patterns were visualized on 1.2% w/v agarose gels illuminated with UV light following ethidium bromide staining. As shown in Figure 1, the band patterns were identical for 8127, 8140, and 8152 with each of the three primers used, and distinct from those of ATCC 2001, confirming the isogenicity of the isolates collected from the patient.
Figure 1.
RAPD gel patterns for C. glabrata isolates 8127, 8140, 8152 and ATCC 2001 (lanes 2–5, in order) obtained using primers OPA-18 (panel A), OPE-18 (panel B), and AP50-1 (panel C). Lane 1 contains 1 kb DNA ladder in panels A, B and C. DNA shown is within the 0.5–4 kb region of the ladder.
To test for upregulation of genes associated with resistance in C. glabrata, relative gene expression was measured in triplicate before and after in vitro exposure to fluconazole. Strains were adjusted to a starting inoculum of ~1 × 104 cells/mL and incubated at 37°C in YPD with shaking to ~1 × 106 cells/mL, after which they were exposed to control (sterile water) or fluconazole 64 µg/mL at 37°C for an additional 12 hours. Cells were harvested by centrifugation and total RNA extracted using the Yeastar RNA Kit (Zymo Research Corp., Orange, CA). Reverse transcription to cDNA was performed (GeneAmp RNA PCR Kit; Applied Biosystems, Inc., Foster City, CA). Relative gene expression was determined by real-time PCR (ABI PRISM 7300 Sequence Detection System) with primers and Taqman probes (TAMRA as the 3’ quencher dye) specific for DNA encoding the C. glabrata efflux pumps CDR1 and PDH1, and the transcriptional regulator PDR1. ACT1 served as the housekeeping gene and was amplified in separate PCR reactions. Relative gene expression levels were calculated by the 2−ΔΔCT method [8]. Differences in gene expression levels between cells exposed to fluconazole and control were compared using one-way analysis of variance (ANOVA) with Tukey’s post-test for multiple comparisons. A p-value of ≤ 0.05 was considered significant.
RESULTS AND DISCUSSION
For isolate 8127 recovered during fluconazole prophylaxis, the transcription levels of CDR1, PDH1, and PDR1 were elevated without in vitro exposure to this azole (≥ 2.25-fold compared to ATCC 2001; Figure 2A). However, the expression of these genes was not further increased upon exposure to fluconazole. These results suggest constitutive expression of these genes affecting fluconazole susceptibility within this isolate. In contrast, in vitro exposure to fluconazole increased the expression of these genes in the isolates recovered from the patient’s oral wash following the discontinuation of azole prophylaxis (Figure 2B and C). Transcription levels of CDR1, PDR1, and PDH1 were significantly increased in isolate 8152 (7.0 ± 2.7, 3.6 ± 0.05, and 2.7 ± 0.4 fold, respectively; p < 0.05) following exposure to fluconazole. In isolate 8140, CDR1 transcription also increased 4.0 ± 0.8 fold following exposure to fluconazole, although this did not reach statistical significance. These data suggest a transient and reversible increase in the expression of these genes affecting fluconazole activity in the isolates recovered after the discontinuation of fluconazole.
Figure 2.
Relative gene expression of CDR1, PDR1, and PDH1 in isolates (A) 8127, (B) 8140, and (C) 8152 relative to ATCC 2001 in the presence and absence of fluconazole (FLC). Fluconazole exposure occurred at a concentration of 64 µg/mL for 12 hours. Expression levels were normalized using ACT1 as the housekeeping gene.
To our knowledge, this is the first case report describing rapid loss of fluconazole resistance following discontinuation of this agent. Following detection of the C. glabrata isolate resistant to fluconazole, a clinical decision was made to change antifungal prophylaxis to an echinocandin. Although it is unknown if this switch in prophylaxis regimens resulted in the rapid loss of fluconazole resistance, the rapid decrease in MICs to this azole after only 6 days is impressive. This loss of resistance is in contrast to previous reports in which resistance develops over time and has been attributed to gain of function mutations within the transcription factor PDR1 [9]. Whether this occurred in our patient cannot be determined as isolates prior to fluconazole prophylaxis azole are not available. It is unknown if this loss of resistance is clinically significant as the patient was colonized but without oropharyngeal candidiasis. Furthermore, it is unknown if fluconazole could be used for subsequent therapy in this patient. However, this may be problematic as in vitro exposure to fluconazole rapidly increased resistant gene expression in these susceptible isolates. Any future use of fluconazole in such a patient should include antifungal susceptibility monitoring.
Table 1.
Primer sequences used for RAPD and gene expression of CDR1, PDR1, PDH1, and ACT1 in C. glabrata isolates. (GenBank Accession Numbers)
| Primer | Sequence |
|---|---|
| RAPD | |
| AP50-1 | 5’-GATTCAGACC |
| OPA-18 | 5’-AGCTGACCGT |
| OPE-18 | 5’-GGACTGCAGA |
|
CDR1 (AF109723) |
Forward 5’-CAAACCATACTCCCTTGGCTGTTA |
| Reverse 5’-GAAGTTGGCCTGGTATTCGATATC | |
| Probe 5’-FAM-GAAGTTGGCCTGGTATTCGATATC-TAMRA-3’ | |
|
PDR1 (AY700584) |
Forward 5’-TCGGCGAGGGTAAATTCAAC |
| Reverse 5’-CCAACTGCGTTTGATTCCTTAAG | |
| Probe 5’-FAM-TACACTAACTGCATCTCCCTTATCGG-TAMRA-3’ | |
|
PDH1 (AF046120) |
Forward 5’-TGGGCAACATGCCAACTG |
| Reverse 5’-AGGTTGGTGAATAGTGCATAAGATTG | |
| Probe 5’-FAM-TGAAGAAACTGGCAAACCACGGACA-TAMRA-3’ | |
| ACT1 | Forward 5’-AATTGAGAGTCGCCCCAGAA |
| Reverse 5’-CTGTTAGACTTTGGGTTCATTGGA | |
| Probe 5’-FAM-ACACCCAGTCTTGTTGACCGAGG-TAMRA-3’ | |
ACKNOWLEDGMENTS
This work was support in part by Public Health Service Grant DE-18096 from the National Institute of Dental and Craniofacial Research. The authors gratefully acknowledge Jon Maust and Marcos Olivo for their technical assistance with yeast identification and isolation.
Footnotes
TRANSPARENCY DECLARATION
SDW, ACV, SK, SB, SAL, WRK, JJT, COF: None to declare N.P.W. has received research support from Pfizer, Schering-Plough, and CyDex Pharmaceuticals. T.F.P. has received research support from Merck, Pfizer, Schering-Plough, and Nektar Therapeutics, has served on the speakers bureau for Merck and Pfizer, and as a consultant for Basilea, Merck, Nektar, Pfizer, and Toyama. S.W.R. has received research support from Pfizer, Schering-Plough, and Astellas
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