Analysis of the transport functions of individual Candida albicans plasma membrane drug efflux pumps is hampered by the multitude of endogenous transporters. We have stably expressed C. albicans Cdr1p, the major pump implicated in multiple-drug-resistance phenotypes, from the genomic PDR5 locus in a Saccharomyces cerevisiae mutant (AD1-8u ؊ ) from which seven major transporters of the ATP-binding cassette (ABC) family have been deleted. High-level expression of Cdr1p, under the control of the S. cerevisiae PDR5 promoter and driven by S. cerevisiae Pdr1p transcriptional regulator mutation pdr1-3, was demonstrated by increased levels of mRNA transcription, increased levels of nucleoside triphosphatase activity, and immunodetection in plasma membrane fractions. S. cerevisiae AD1-8u؊ was hypersensitive to azole antifungals (the MICs at which 80% of cells were inhibited [MIC 80 s] were 0.625 g/ml for fluconazole, <0.016 g/ml for ketoconazole, and <0.016 g/ml for itraconazole), whereas the strain (AD1002) that overexpressed C. albicans Cdr1p was resistant to azoles (MIC 80 s of fluconazole, ketoconazole, and itraconazole, 30, 0.5, and 4 g/ml, respectively). Drug resistance correlated with energy-dependent drug efflux. AD1002 demonstrated resistance to a variety of structurally unrelated chemicals which are potential drug pump substrates. The controlled overexpression of C. albicans Cdr1p in an S. cerevisiae background deficient in other pumps allows the functional analysis of pumping specificity and mechanisms of a major ABC transporter involved in drug efflux from an important human pathogen.Candida albicans is an asexual diploid fungus that causes opportunistic infections commonly seen in immunocompromised and debilitated patients (9, 30). An estimated 33 to 55% of patients with human immunodeficiency virus infection and AIDS contract oropharyngeal candidosis (34), and the synthetic triazole fluconazole has been the mainstay of their treatment. The widespread use of prolonged fluconazole therapy has increased the incidence of treatment failure due to fluconazole-resistant C. albicans (3,14,21,34,42). A number of studies have identified the major azole resistance mechanisms (1,20,38,41,42,(44)(45)(46). These include overexpression of, or mutations in, the drug target, 14␣-sterol demethylase; mutations in other parts of the sterol biosynthesis pathway; and, most commonly, overexpression of drug efflux proteins.C. albicans possesses transporters such as Cdr1p and Cdr2p with homology to proteins of the ATP-binding cassette (ABC) family (10,16,18,19,31), as well as Ben r p, which has homology to the major facilitator superfamily (MFS) class of drugproton antiport efflux pumps (1,5,36,46). The BEN r gene encodes a transporter associated with resistance to benomyl and methotrexate when it is expressed in Saccharomyces cerevisiae. The C. albicans CDR1 gene is a homologue of S. cerevisiae PDR5, which encodes a multidrug efflux pump, and CDR1 is the gene most often associated with energy-dependent drug efflux in fluconazole-r...

Most Candida krusei strains are innately resistant to fluconazole (FLC) and can cause breakthrough candidemia in immunocompromised individuals receiving long-term prophylactic FLC treatment. Although the azole drug target, Erg11p, of C. krusei has a relatively low affinity for FLC, drug efflux pumps are also believed to be involved in its innate FLC resistance. We describe here the isolation and characterization of Abc1p, a constitutively expressed multidrug efflux pump, and investigate ERG11 and ABC1 expression in C. krusei. Examination of the ERG11 promoter revealed a conserved azole responsive element that has been shown to be necessary for the transcription factor Upc2p mediated upregulation by azoles in related yeast. Extensive cloning and sequencing identified three distinct ERG11 alleles in one of two C. krusei strains. Functional overexpression of ERG11 and ABC1 in Saccharomyces cerevisiae conferred high levels of resistance to azoles and a range of unrelated Abc1p pump substrates, while small molecule inhibitors of Abc1p chemosensitized C. krusei to azole antifungals. Our data show that despite the presence of multiple alleles of ERG11 in some, likely aneuploid, C. krusei strains, it is mainly the low affinity of Erg11p for FLC, together with the constitutive but low level of expression of the multidrug efflux pump Abc1p, that are responsible for the innate FLC resistance of C. krusei.

ObjectivesThe Mycoses Forum in Japan has developed management bundles for candidaemia to incorporate into bedside practice. The aim of this study was to investigate nationwide compliance with the bundles and their impact on clinical outcomes.MethodsNon-neutropenic patients treated with antifungals for candidaemia were surveyed. Bundles consist of nine items to complete. Data were sent to the central office between July 2011 and April 2012.ResultsSix hundred and eight patients were analysed. The compliance rate for achieving all elements was 6.9%, and it increased to 21.4% when compliance was analysed by the bundle except for oral switch. There was a significant difference in clinical success between patients with and without compliance [92.9% versus 75.8% (P = 0.011)]. Compliance with the bundles, however, failed to be an independent factor associated with favourable outcomes. When step-down oral therapy was excluded from the elements of compliance, compliance with the bundles was revealed to be an independent predictor of clinical success (OR 4.42, 95% CI 2.05–9.52) and mortality (OR 0.27, 95% CI 0.13–0.57). Independent individual elements contributing to clinical success were removal of central venous catheters within 24 h, assessment of clinical efficacy on the third to the fifth day and at least 2 weeks of therapy after clearance of candidaemia.ConclusionsCompliance with the bundles for candidaemia had a beneficial effect on clinical outcomes. Promotion of the bundles approach may have the potential to narrow the gap between clinical evidence and bedside practice.

These studies demonstrated that LFO is safe when administered once daily up to 1200 mg/day. This is the first report on the safety of licorice flavonoids in an oil preparation and the first report on the pharmacokinetics of glabridin in human subjects.