Candida glabrata has emerged as a common cause of fungal infection. This yeast has intrinsically low susceptibility to azole antifungals such as fluconazole, and mutation to frank azole resistance during treatment has been documented. Potential resistance mechanisms include changes in expression or sequence of ERG11 encoding the azole target. Alternatively, resistance could result from upregulated expression of multidrug transporter genes; in C. glabrata these include CDR1 and PDH1. By RNA hybridization, 10 of 12 azole-resistant clinical isolates showed 6-to 15-fold upregulation of CDR1 compared to susceptible strains. In 4 of these 10 isolates PDH1 was similarly upregulated, and in the remainder it was upregulated three-to fivefold, while ERG11 expression was minimally changed. Laboratory mutants were selected on fluconazole-containing medium with glycerol as carbon source (to eliminate mitochondrial mutants). Similar to the clinical isolates, six of seven laboratory mutants showed unchanged ERG11 expression but coordinate CDR1-PDH1 upregulation ranging from 2-to 20-fold. Effects of antifungal treatment on gene expression in susceptible C. glabrata strains were also studied: azole exposure induced CDR1-PDH1 expression 4-to 12-fold. These findings suggest that these transporter genes are regulated by a common mechanism. In support of this, a mutation associated with laboratory resistance was identified in the C. glabrata homolog of PDR1 which encodes a regulator of multidrug transporter genes in Saccharomyces cerevisiae. The mutation falls within a putative activation domain and was associated with PDR1 autoupregulation. Additional regulatory factors remain to be identified, as indicated by the lack of PDR1 mutation in a clinical isolate with coordinately upregulated CDR1-PDH1.

SummaryCandida glabrata emerged in the last decade as a common cause of mucosal and invasive fungal infection, in large part due to its intrinsic or acquired resistance to azole antifungals such as fluconazole. In C. glabrata clinical isolates, the predominant mechanism behind azole resistance is upregulated expression of multidrug transporter genes CDR1 and PDH1. We previously reported that azole-resistant mutants (MIC Ն 64 mg ml ) to both F15 and 66032 and eliminated both constitutive and fluconazole-induced CDR1-PDH1 expression. Reintroduction of wild-type or F15 PDR1 fully reversed these effects; together these results demonstrate a role for this gene in both acquired and intrinsic azole resistance. CDR1 disruption had a partial effect, reducing fluconazole trailing in both strains while restoring wild-type susceptibility (MIC = 16 mg ml -1 ) to F15. In an azole-resistant clinical isolate, PDR1 disruption reduced azole MICs eight-to 64-fold with no effect on sensitivity to other antifungals. To extend this analysis, C. glabrata microarrays were generated and used to analyse genome-wide expression in F15 relative to its parent. Homologues of 10 S. cerevisiae genes previously shown to be Pdr1-Pdr3 targets were upregulated (YOR1, RTA1, RSB1, RPN4, YLR346c and YMR102c along with CDR1, PDH1 and PDR1 itself) or downregulated (PDR12); roles for these genes include small molecule transport and transcriptional regulation. However, expression of 99 additional genes was specifically altered in C. glabrata F15; their roles include transport (e.g. QDR2, YBT1), lipid metabolism (ATF2, ARE1), cell stress (HSP12, CTA1), DNA repair (YIM1, MEC3) and cell wall function (MKC7, MNT3). These azole resistance-associated changes could affect C. glabrata tissue-specific virulence; in support of this, we detected differences in F15 oxidant, alcohol and weak acid sensitivities. C. glabrata provides a promising model for studying the genetic basis of multidrug resistance and its impact on virulence.

A retrospective survey of 93,775 samples testing positive in Candida species-specific PCR tests performed on cervicovaginal swabs over a 4-year period demonstrated consistent yearly distributions of Candida albicans (89%), C. glabrata (7.9%), C. parapsilosis (1.7%), and C. tropicalis (1.4%). However, the species distributions among different age groups revealed increases in the percentages of non-albicans species with increases in age.Vulvovaginal candidiasis (VVC) is a common fungal infection that affects healthy women of all ages. At least 75% of women will develop one or more infections once during their lifetime, with 5 to 8% of those individuals developing recurrent infections (5, 7). Current literature examining the species distribution of Candida isolates involved in VVC is limited; however, several important observations have been made. For example, one study shows that Candida albicans accounts for 70 to 90% of all VVC cases, with a recent emergence of nonalbicans species (10). The rise in VVC infections, more specifically in those caused by non-albicans species, could be due to several factors, ranging from an increase in over-the-counter antifungal use to an increase in high-risk patient populations (i.e., diabetics and menopausal women). Candida glabrata is the primary non-albicans species emerging in VVC, accounting for up to 14% of infections in immune-competent women (9, 10).In addition to an increase in non-albicans species overall, it is becoming clear that certain patient populations may experience higher risks of infection from these non-albicans species, often leading to limited treatment options. Interestingly, in a few small studies, C. glabrata was found to be the primary species isolated from diabetic (61.3%) and elderly (51.2%) patients with VVC (2, 4, 6, 11). Often, these non-albicans species are associated with elevated MIC levels for the azoles, the most commonly prescribed class of antifungal drugs. It has been well documented that C. glabrata demonstrates both intrinsically low susceptibility to the azoles and the ability to develop frank resistance (8,12,13,14,15,16). Moreover, a recent increase in the trailing phenotype, with low-level resistance to the azoles, has been observed for the Candida tropicalis isolates (1, 3). This highlights the importance of identifying Candida species within clinical samples in order to provide physicians with information concerning the proper treatment for their patients.

dCandida glabrata, the second most common cause of Candida infections, is associated with high rates of mortality and often exhibits resistance to the azole class of antifungal agents. Upc2 and Ecm22 in Saccharomyces cerevisiae and Upc2 in Candida albicans are the transcriptional regulators of ERG11, the gene encoding the target of azoles in the ergosterol biosynthesis pathway. Recently two homologs for these transcription factors, UPC2A and UPC2B, were identified in C. glabrata. One of these, UPC2A, was shown to influence azole susceptibility. We hypothesized that due to the global role for Upc2 in sterol biosynthesis in S. cerevisiae and C. albicans, disruption of UPC2A would enhance the activity of fluconazole in both azole-susceptible dosedependent (SDD) and -resistant C. glabrata clinical isolates. To test this hypothesis, we constructed mutants with disruptions in UPC2A and UPC2B alone and in combination in a matched pair of clinical azole-SDD and -resistant isolates. Disruption of UPC2A in both the SDD and resistant isolates resulted in increased susceptibility to sterol biosynthesis inhibitors, including a reduction in fluconazole MIC and minimum fungicidal concentration, enhanced azole activity by time-kill analysis, a decrease in ergosterol content, and downregulation of baseline and inducible expression of several sterol biosynthesis genes. Our results indicate that Upc2A is a key regulator of ergosterol biosynthesis and is essential for resistance to sterol biosynthesis inhibitors in C. glabrata. Therefore, the UPC2A pathway may represent a potential cotherapeutic target for enhancing azole activity against this organism.

PCR product-based gene disruption has greatly accelerated molecular analysis of Saccharomyces cerevisiae. This approach involves amplification of a marker gene (e.g., URA3) including its flanking regulatory (promoter and polyadenylation) regions using primers that include at their 5' ends about 50 bases of homology to the targeted gene. Unfortunately, this approach has proved less useful in organisms with higher rates of non-homologous recombination; e.g., in the yeast Candida glabrata, desired recombinants represent < or =2% of transformants. We modified the PCR-based approach by eliminating marker-flanking regions and precisely targeting recombination such that marker expression depends on the regulatory sequences of the disrupted gene. Application of this promoter-dependent disruption of genes (PRODIGE) method to three C. glabrata genes (SLT2, LEM3, and PDR1) yielded desired recombinants at frequencies of 20, 31, and 11%, the latter representing a weakly expressed gene. For Candida albicans LEM3 and RHO1, specificity was 79-95% for one or both alleles, >sixfold higher than the published results with conventional PCR-based gene disruption. All 5 C. glabrata and C. albicans mutants had predicted phenotypes of calcofluor hypersensitivity (slt2Delta and RHO1/rho1Delta), cycloheximide hypersensitivity (pdr1Delta), or miltefosine resistance (lem3Delta and lem3Delta/lem3Delta). PRODIGE application to the S. cerevisiae PDR5 gene in strains with and without the Pdr1-Pdr3 transcriptional activators of this gene confirmed that transformant yield and growth rate depend on promoter strength. Using this PDR5 promoter-URA3 recombinant, we further demonstrate a simple extension of the method that yields regulatory mutants via 5-fluoroorotic acid selection. PRODIGE warrants testing in other yeast, molds, and beyond.

In addition to transcriptional regulation of Cdr1p, this study identified the differential expression of several proteins that may contribute to azole resistance and suggests the possibility for a post-transcriptional mechanism for increased expression of Erg11p.

The opportunistic yeast Candida glabrata is increasingly refractory to antifungal treatment or prophylaxis and relatedly is increasingly implicated in health care-associated infections. To elucidate the epidemiology of these infections, strain typing is required. Sequence-based typing provides multiple advantages over length-based methods, such as pulsed-field gel electrophoresis (PFGE); however, conventional multilocus sequence typing (targeting 6 conserved loci) and whole-genome sequencing are impractical for routine use. A commercial sequence-based typing service for C. glabrata that targets polymorphic tandem repeatcontaining loci has recently been developed. These CgMT-J and CgMT-M services were evaluated with 56 epidemiologically unrelated isolates, 4 to 7 fluconazole-susceptible or fluconazole-resistant isolates from each of 5 center A patients, 5 matched pairs of fluconazole-susceptible/resistant isolates from center B patients, and 7 isolates from a center C patient who responded to then failed caspofungin therapy. CgMT-J and CgMT-M generated congruent results, resolving isolates into 24 and 20 alleles, respectively. Isolates from all but one of the center A patients shared the same otherwise rare alleles, suggesting nosocomial transmission. Unexpectedly, Pdr1 sequencing showed that resistance arose independently in each patient. Similarly, most isolates from center B also clustered together; however, this may reflect a dominant clone since their alleles were shared by multiple unrelated isolates. Although distinguishable by their echinocandin susceptibilities, all isolates from the center C patient shared alleles, in agreement with the previously reported relatedness of these isolates based on PFGE. Finally, we show how phylogenetic clusters can be used to provide surrogate parents to analyze the mutational basis for antifungal resistance.

A multiplex quantitative reverse transcription-PCR assay was developed to detect azole resistance in Candida glabrata, an important opportunistic pathogen that develops resistance rapidly. Resistance was defined as a >3-fold increase in CDR1 expression by this assay, which proved to be 100% sensitive and 95% specific in comparison to the gold standard broth microdilution assay.