A Combination Fluorescence Assay Demonstrates Increased Efflux Pump Activity as a Resistance Mechanism in Azole-Resistant Vaginal Candida albicans Isolates

Abstract

Candida albicans is a pathogenic fungus causing vulvovaginal candidiasis (VVC). Azole drugs, such as fluconazole, are the most common treatment for these infections. Recently, azole-resistant vaginal C. albicans isolates have been detected in patients with recurring and refractory vaginal infections. However, the mechanisms of resistance in vaginal C. albicans isolates have not been studied in detail. In oral and systemic resistant isolates, overexpression of the ABC transporters Cdr1p and Cdr2p and the major facilitator transporter Mdr1p is associated with resistance. Sixteen fluconazole-susceptible and 22 fluconazole-resistant vaginal C. albicans isolates were obtained, including six matched sets containing a susceptible and a resistant isolate, from individual patients. Using quantitative real-time reverse transcriptase PCR (qRT-PCR), 16 of 22 resistant isolates showed overexpression of at least one efflux pump gene, while only 1 of 16 susceptible isolates showed such overexpression. To evaluate the pump activity associated with overexpression, an assay that combined data from two separate fluorescent assays using rhodamine 6G and alanine β-naphthylamide was developed. The qRT-PCR results and activity assay results were in good agreement. This combination of two fluorescent assays can be used to study efflux pumps as resistance mechanisms in clinical isolates. These results demonstrate that efflux pumps are a significant resistance mechanism in vaginal C. albicans isolates.

FIG 1

Gene expression levels of all 38 vaginal isolates. (a to d) Expression levels of mRNA for the genes CDR1 (a), CDR2 (b), MDR1 (c), and ERG11 (d). The data are normalized to those for wild-type strain SC5314. ACT1 was used as an expression control. Gene expression is plotted on a log scale as colored bars, and the error bars for gene expression represent the standard errors from triplicate assays. The strains on the x axis are arranged by increasing FLC MICs (line graph). The dotted horizontal lines in panels a to d signify 2-fold upregulation or 2-fold downregulation of mRNA expression compared to that in SC5314 (wild type).

FIG 2

Gene expression levels between matched isolates. The expression of the CDR1, CDR2, MDR1, and ERG11 genes in matched resistant isolates (isolates S2, S4, S8, and S12) is normalized to that in their susceptible partner (S1, S3, S7, and S11, respectively). Gene expression is plotted on a log scale, and error bars represent the standard errors from triplicate assays. Dotted horizontal lines signify 2-fold upregulation or 2-fold downregulation in mRNA expression compared to that in the matched partner. The comparison of expression in S6 and S5 is not shown because the MICs for these isolates did not change.

FIG 3

Efflux map of S. cerevisiae strains expressing efflux pumps. (Inset) Illustration of how the values are graphed. The R6G values (y axis) represent the ratio of the R6G efflux slope in the test strain compared to that in the control (strain ADΔ). The Ala-Nap values (x axis) represent the ratio of Ala-Nap retention in the test strain compared to that in the control. Retention is measured as the difference in Ala-Nap retention in the presence of glucose and Ala-Nap retention in the absence of glucose. A high level of Ala-Nap retention indicates a low level of efflux, and a low level of retention represents a high level of efflux. The negative control for Fig. 3 is ADΔ. Strains AD-CDR1, AD-CDR2, and AD-MDR1 express, respectively, CDR1, CDR2, and MDR1. S288C expresses PDR5, a homolog of the CDRs. Error bars and slopes were calculated by using the LINEST function in Microsoft Excel software on curves generated from biological triplicates for both the Ala-Nap and R6G assays.

FIG 4

Efflux map of 38 vaginal C. albicans clinical isolates. The controls used were strains 2-76, 12-99, SC5314, and DSY1050 (black circles). The data were normalized to those for strain 2-76. Values were calculated as described in the inset in Fig. 3. Green symbols, FLC-susceptible strains; red symbols, FLC-resistant strains; yellow triangles, FLC resistant-strains overexpressing ERG11; red squares, isolates S14, S36, and S37, which are unusual in showing R6G efflux but not Ala-Nap efflux; green squares, isolates S24 and S25, which are unusual as they are susceptible isolates showing significant efflux by both the Ala-Nap and R6G assays, most likely due to CDR4 overexpression. The results in the region between the vertical dotted lines are not significantly different from those for the control for the Ala-Nap assay, and the results in the region between the horizontal dotted lines are not significantly different from those for the control for the R6G assay. Significance was calculated using one-way analysis of variance with Dunnett's multiple-comparison tests. Error bars and slopes were calculated by using the LINEST function in Microsoft Excel software on curves generated from biological triplicates from both the Ala-Nap and R6G assays.

FIG 5

Efflux map of matched vaginal C. albicans clinical isolates. Strains 2-76 and 12-99 were used as efflux controls, and the data are normalized to those for strain 2-76. Calculations were performed as described in the inset in Fig. 3. Green circles, FLC-susceptible strains; red circles, FLC-resistant strains; yellow triangles, FLC-resistant strains overexpressing ERG11. Error bars and slopes were calculated as described in Materials and Methods. Isolate S5 and S6 are both resistant.