Mechanisms of azole resistance in clinical isolates of Candida glabrata collected during a hospital survey of antifungal resistance

Abstract

The increasing use of azole antifungals for the treatment of mucosal and 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, which 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-year 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 fluconazole-resistant 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-resistant 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 the 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 isolate, 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 of 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 evidence that the upregulation of the CgCDR1-, CgCDR2-, and CgSNQ2-encoded efflux pumps might explain the azole resistance in our set of isolates.

FIG. 1.

Distributions of percentages of C. glabrata isolates susceptible to four azole antifungal agents among 267 clinical isolates. S, the isolates are susceptible; S-DD, the isolates are susceptible in a dose-dependent manner; R, the isolates are resistant. The azole susceptibility categories were determined as described in the text.

FIG. 2.

Expression of CgCDR1, CgCDR2, and CgSNQ2 in the 25 unmatched C. glabrata isolates, as determined by real-time RT-PCR analysis (see the text for details). For each target gene, the relative amount of transcription (i.e., in reference to that by susceptible control isolate DSY562) was compared to that of URA3. The results are reported as the log₁₀ fold increases in gene expression levels. Error bars show standard deviations. The x axis indicates the 25 isolates, arranged as resistant (R) and susceptible dose dependent (S-DD) to fluconazole.

FIG. 3.

Expression of CgCDR1, CgCDR2, and CgSNQ2 in matched susceptible and resistant isolates from four patients, as determined by real-time RT-PCR analysis (see the text for details). For each target gene, the relative amount of transcription (i.e., in reference to that by susceptible control isolate DSY562) was compared to that of the URA3. The results are reported as the log₁₀ fold increases in gene expression levels. Error bars show standard deviations.