Biomedical subjects

D J Sullivan

Publications and source records attributed to D J Sullivan.

At least 19 recordsLinked to original sources

PubMed2002

Candida dubliniensis candidaemia in an HIV-positive patient in Ireland.

Candida dubliniensis was first identified in Dublin in 1995 in oral isolates recovered from human immunodeficiency virus (HIV)-infected individuals. Although C. dubliniensis has been primarily recovered from the oral cavities of HIV-infected individuals, the number of reports describing its isolation from HIV-negative individuals, including cases of candidaemia, is growing. To date there has only been one report of C. dubliniensis candidaemia in an HIV-infected patient, in this case from the USA. In the present study, 2 Candida isolates recovered from blood samples were presumptively identified as C. dubliniensis on the basis of their dark green coloration on CHROMagar Candida medium and lack of growth at 45 degrees C. This identification was confirmed by carbohydrate assimilation profile analysis and by polymerase chain reaction (PCR) analysis with C. dubliniensis-specific PCR primers. Both isolates were susceptible to fluconazole. The isolates were found retrospectively to be from a single HIV-infected patient who was receiving broad-spectrum antibacterials at the time of isolation of C. dubliniensis from blood. This study represents the first documented case of C. dubliniensis bloodstream infection in Ireland and is only the second case of C. dubliniensis bloodstream infection identified in an HIV-infected individual anywhere in the world.

Adult↗

PubMed2001

Haemoproteus and Schistosoma synthesize heme polymers similar to Plasmodium hemozoin and beta-hematin.

Many parasites digest hemoglobin as an amino acid source, but only a few produce heme polymer pigment instead of catabolizing heme via heme oxygenase. This work compares purified heme polymers produced by Haemoproteus columbae and Schistosoma mansoni to that of Plasmodium falciparum hemozoin and synthetic beta-hematin. Fourier-transform infrared spectroscopy identifies the signature peaks of the common iron-carboxylate bond characteristic in all four heme polymers. However, all pigments could be distinguished by quite different three-dimensional structure visualized by Field Emission Inlens Scanning Electron Microscopy. Both P. falciparum and H. columbae heme polymers had a symmetrical shape unlike the amorphous S. mansoni heme polymer and beta-hematin. All four heme pigments serve as templates for heme polymer extension, which was inhibitable by chloroquine and other quinoline antimalarials. The polymers showed different levels of resistance to hydrogen peroxide degradation. This work identifies another genus, Haemoproteus, capable of intracellular heme polymer formation. The different three-dimensional structures of each pigment implicate genus specific formation of heme polymer, variation of inhibition of polymer extension by the quinolines and degradation by hydrogen peroxide.

Animals↗

PubMed2001

MDR1-mediated drug resistance in Candida dubliniensis.

Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans. Candida dubliniensis readily develops resistance to the azole antifungal agent fluconazole, both in vitro and in infected patients, and this resistance is usually associated with upregulation of the CdMDR1 gene, encoding a multidrug efflux pump of the major facilitator superfamily. To determine the role of CdMDR1 in drug resistance in C. dubliniensis, we constructed an mdr1 null mutant from the fluconazole-resistant clinical isolate CM2, which overexpressed the CdMDR1 gene. Sequential deletion of both CdMDR1 alleles was performed by the MPA(R)-flipping method, which is based on the repeated use of a dominant mycophenolic acid resistance marker for selection of integrative transformants and its subsequent deletion from the genome by FLP-mediated, site-specific recombination. In comparison with its parental strain, the mdr1 mutant showed decreased resistance to fluconazole but not to the related drug ketoconazole. In addition, we found that CdMDR1 confers resistance to the structurally unrelated drugs 4-nitroquinoline-N-oxide, cerulenin, and brefeldin A, since the enhanced resistance to these compounds of the parent strain CM2 compared with the matched susceptible isolate CM1 was abolished in the mdr1 mutant. In contrast, CdMDR1 inactivation did not cause increased susceptibility to amorolfine, terbinafine, fluphenazine, and benomyl, although overexpression of CdMDR1 in a hypersusceptible Saccharomyces cerevisiae strain had previously been shown to confer resistance to these compounds. The effect of CdMDR1 inactivation was identical to that seen in two similarly constructed C. albicans mdr1 mutants. Therefore, despite species-specific differences in the amino acid sequences of the Mdr1 proteins, overexpression of CaMDR1 and CdMDR1 in clinical C. albicans and C. dubliniensis strains seems to confer the same drug resistance profile in both species.

Antifungal Agents↗

PubMed2001

Isogenic strain construction and gene targeting in Candida dubliniensis.

Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans but differs from it with respect to epidemiology, certain virulence characteristics, and the ability to develop fluconazole resistance in vitro. A comparison of C. albicans and C. dubliniensis at the molecular level should therefore provide clues about the mechanisms used by these two species to adapt to their human host. In contrast to C. albicans, no auxotrophic C. dubliniensis strains are available for genetic manipulations. Therefore, we constructed homozygous ura3 mutants from a C. dubliniensis wild-type isolate by targeted gene deletion. The two URA3 alleles were sequentially inactivated using the MPA(R)-flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. The URA3 gene from C. albicans (CaURA3) was then used as a selection marker for targeted integration of a fusion between the C. dubliniensis MDR1 (CdMDR1) promoter and a C. albicans-adapted GFP reporter gene. Uridine-prototrophic transformants were obtained with high frequency, and all transformants of two independent ura3-negative parent strains had correctly integrated the reporter gene fusion into the CdMDR1 locus, demonstrating that the CaURA3 gene can be used for efficient and specific targeting of recombinant DNA into the C. dubliniensis genome. Transformants carrying the reporter gene fusion did not exhibit detectable fluorescence during growth in yeast extract-peptone-dextrose medium in vitro, suggesting that CdMDR1 is not significantly expressed under these conditions. Fluconazole had no effect on MDR1 expression, but the addition of the drug benomyl strongly activated the reporter gene fusion in a dose-dependent fashion, demonstrating that the CdMDR1 gene, which encodes an efflux pump mediating resistance to toxic compounds, is induced by the presence of certain drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Plasmodium falciparum: histidine-rich protein II is expressed during gametocyte development.

Both early gametocytes (I-II) and asexual trophozoite stages of Plasmodium falciparum digest hemoglobin and detoxify haem by polymerizing it into parasite pigment called hemozoin. The mechanism of polymerization is unclear but it has been proposed that histidine-rich protein II may facilitate transport of hemoglobin to the food vacuole and catalyze the polymerization in asexual stages. We describe the transcription of histidine-rich protein II in gametocytes by Northern blot analysis and the expression of the protein in these stages by immunoprecipitation and Western blotting. Localization of histidine-rich protein II within the gametocyte by immunofluorescence assay and immunoelectron microscopy clearly illustrated the presence of this molecule in the infected red cell cytosol in the early stages of gametocyte development and internalization in the later gametocyte as it matures. There is a strong correlation between the stage-specific trafficking of histidine-rich protein II in gametocytes and the susceptibility of early but not late gametocytes to the antimalarial drug chloroquine.

Animals↗

PubMed2000

Termination of life support after major trauma.

As the population continues to age, greater numbers and more severely injured elderly patients require care in ICUs. With the attendant increase in the medical complexity of such patients, investigators anticipate that trauma and critical care resources will become increasingly stretched. Because of economic and societal forces, it will become increasingly important for trauma surgeons to appropriately counsel patients and their families regarding the outcome from their injuries and to become comfortable approaching families about withdrawal of support when medical futility is recognized. The authors propose the following guidelines for discussing limitation or termination of life support with patients and their families. Physicians should (1) discuss the patient's wishes regarding life support on admission or early in the hospital course; (2) at the initial discussion, establish who the decision maker will be if the patient is or becomes incapacitated; (3) maintain regular communication and continuity of care; and (4) inevitably, when conflict occurs, involve consultants and a hospital ethics committee for assistance in its resolution.

Advance Directives↗

PubMed2000

Isolation of C. dubliniensis from insulin-using diabetes mellitus patients.

The identification of the novel candidal species, C. dubliniensis, from oral swab studies of HIV-seropositive and -seronegative individuals has led to speculation that such a species may also reside in the oral cavity of other patient groups. In this study involvement of the newly described species, C. dubliniensis, was investigated in oral carriage and disease in 414 insulin-using diabetes mellitus patients. Seventy-seven percent of the diabetic patients carried candidal species in the oral cavity. C. albicans was the most commonly identified candidal species. This was followed by C. dubliniensis, which was isolated on 64 occasions. Colonisation with multiple candidal species was common, and C. dubliniensis was present in both carriage and disease states. Seven patients without signs of oral disease had C. dubliniensis isolated as the sole candidal species, while the same species was associated with various forms of oral candidosis in six patients.

Candida↗

PubMed2000

Muscle force production and functional performance in spastic cerebral palsy: relationship of cocontraction.

OBJECTIVE: To determine cocontraction's relation to strength and motor function in children with spastic cerebral palsy (CP). DESIGN: Prospective evaluation with a convenience sample of 10 subjects. SETTING: Pediatric rehabilitation center at a tertiary care hospital. PATIENTS: Ten ambulatory children with spastic CP, mean age 5 to 14yrs. MAIN OUTCOME MEASURES: A single comprehensive assessment of hamstring and quadriceps muscle strength; gait analysis while monitoring electromyographic (EMG) activity in those muscles; administration of the Gross Motor Function Measure (GMFM); heart-rate monitoring during quiet rest versus gait to compute an energy expenditure index (EEI). Cocontraction ratios and magnitudes were determined for the gait and strength testing trials using the EMG data. RESULTS: Cocontraction ratios during strength tests correlated directly with those during free gait. Cocontraction magnitude and total EMG magnitude had an inverse relationship to EEI; children with more muscle activity in the agonist and antagonist tended to be more energy efficient. Knee extensor muscle strength correlated positively with the GMFM and gait velocity. Neither cocontraction ratio nor magnitude during gait was related to strength. CONCLUSIONS: Children with CP used a similar muscle activation strategy across two different motor tasks. Strength and cocontraction were uniquely related to different aspects of motor function. Further research is needed to quantify more precisely cocontraction and force to EMG relations in this population.

Adolescent↗

PubMed2000

Emerging pathogens.

The ever increasing numbers of immunosuppressed individuals has led to a significant increase in the incidence of opportunistic infections, particularly those caused by fungi. The epidemiology of infections caused by the common fungal pathogens such as Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus has been well documented. However, in addition to these, a number of species which have previously been unrecognized (e.g., C. dubliniensis) or have previously been assumed to be non-pathogenic (e.g., Saccharomyces cerevisiae, Scedosporium spp. and Fusarium spp.) have emerged as agents of human disease. Since these species have only been identified recently as human pathogens, their role in disease is poorly understood. In most cases, identification of these species is problematic and therefore their epidemiology has yet to be elucidated adequately. In addition, several of these species fail to respond to conventional antifungal therapies. In this article, we describe the emergence of two separate yeast species (C. dubliniensis and S. cerevisiae) and two separate groups of moulds (Scedosporium prolificans and Fusarium spp.), as human pathogens. It is apparent from what we already know, that much work has yet to be performed before we have a clear understanding of how these species cause disease and most importantly how they can be controlled.

Candida↗

PubMed2000

New monoclonal antibody specific for Candida albicans germ tube.

Hydrophobic components of the germ tube of the dimorphic pathogenic fungus Candida albicans were used as immunogens to prepare monoclonal antibodies (MAbs). Among the resulting MAbs, one (MAb 16B1-F10) was shown by indirect immunofluorescence to be specific to the surface of the mycelium phase of the C. albicans and C. stellatoidea species. No labeling of any other genera and Candida species tested was observed, including C. dubliniensis, a newly described species which has many phenotypic similarities to C. albicans. This phase-specific epitope resides on a protein moiety. The molecular mass of the antigen released by Zymolyase digestion was determined by gel filtration and ranges from 25 to 166 kDa. The antigen was also shown to be highly hydrophobic. This anti-C. albicans cell wall surface-specific MAb may be a good candidate for use in tests for the rapid differentiation of the two closely related species C. albicans and C. dubliniensis.

Antibodies, Fungal↗

PubMed1999

Rapid identification of Candida dubliniensis with commercial yeast identification systems.

Candida dubliniensis is a newly described species that is closely related phylogenetically to Candida albicans and that is commonly associated with oral candidiasis in human immunodeficiency virus-positive patients. Several recent studies have attempted to elucidate phenotypic and genotypic characteristics of use in separating the two species. However, results obtained with simple phenotypic tests were too variable and tests that provided more definitive data were too complex for routine use in the clinical laboratory setting. The objective of this study was to determine if reproducible identification of C. dubliniensis could be obtained with commercial identification kits. The substrate reactivity profiles of 80 C. dubliniensis isolates were obtained by using the API 20C AUX, ID 32 C, RapID Yeast Plus, VITEK YBC, and VITEK 2 ID-YST systems. The percentages of C. dubliniensis isolates capable of assimilating or hydrolyzing each substrate were compared with the percentages from the C. albicans profiles in each kit's database, and the results were expressed as percent C. dubliniensis and percent C. albicans. Any substrate that showed >50% difference in reactivity was considered useful in differentiating the species. In addition, assimilation of methyl-alpha-D-glucoside (MDG), D-trehalose (TRE), and D-xylose (XYL) by the same isolates was investigated by the traditional procedure of Wickerham and Burton (L. J. Wickerham and K. A. Burton, J. Bacteriol. 56:363-371, 1948). At 48 h (the time recommended by the manufacturer for its new database), we found that the assimilation of four carbohydrates in the API 20C AUX system could be used to distinguish the species, i.e., glycerol (GLY; 88 and 14%), XYL (0 and 88%), MDG (0 and 85%), and TRE (15 and 97%). Similarly, results with the ID 32 C system at 48 h showed that XYL (0 and 98%), MDG (0 and 98%), lactate (LAT; 0 and 96%), and TRE (30 and 96%) could be used to separate the two species. Phosphatase (PHS; 9 and 76%) and alpha-D-glucosidase (23 and 94%) proved to be the most useful for separation of the species in the RapID Yeast Plus system. While at 24 h the profiles obtained with the VITEK YBC system showed that MDG (10 and 95%), XYL (0 and 95%), and GLY (26 and 80%) could be used to separate the two species, at 48 h only XYL (6 and 95%) could be used to separate the two species. The most useful substrates in the VITEK 2 ID-YST system were TRE (1 and 89%), MDG (1 and 99%), LAT (4 and 98%), and PHS (83 and 1%). While the latter kit was not yet commercially available at the time of the study, it would appear to be the most valuable for the identification of C. dubliniensis. Although assimilation of MDG, TRE, and XYL proved to be the most useful for species differentiation by the majority of commercial systems, the results with these carbohydrates by the Wickerham and Burton procedure were essentially the same for both species, albeit following protracted incubation. Thus, it is the rapidity of the assimilation achieved with the commercial systems that allows the differentiation of C. dubliniensis from C. albicans.

AIDS-Related Opportunistic Infections↗

PubMed1999

Identification of Candida dubliniensis based on temperature and utilization of xylose and alpha-methyl-D-glucoside as determined with the API 20C AUX and vitek YBC systems.

To have a better understanding of the role of Candida dubliniensis in clinical infections, it is essential that microbiology laboratories can identify this species rapidly and accurately in clinical specimens. C. dubliniensis has been reported to lack the ability to utilize xylose (XYL) and alpha-methyl-D-glucoside (MDG) and to grow poorly or not at all at 45 degrees C, whereas Candida albicans isolates utilize XYL and MDG and usually grow well at 45 degrees C. We tested 66 isolates of C. dubliniensis and 100 isolates of C. albicans with both the API 20C AUX and Vitek YBC systems to evaluate the ability of the XYL and MDG tests contained within each of these systems to distinguish between the two species. The ability to grow at 45 degrees C was also examined. None of the C. dubliniensis isolates grew at 45 degrees C, and 23 of 100 C. albicans isolates (23%) exhibited poor or no growth at 45 degrees C. The XYL and MDG tests contained within the API 20C AUX system were both negative for all 66 C. dubliniensis isolates and were positive for 98 (XYL) and 56 (MDG) of the 100 C. albicans isolates. With the Vitek system, 64 of 66 C. dubliniensis isolates (97.0%) were XYL negative and 63 (95.0%) were MDG negative. Conversely, 96 of 100 C. albicans isolates (96.0%) were XYL positive and 100 (100.0%) were MDG positive with the Vitek system. Clinical microbiology laboratories could use lack of growth at 45 degrees C and a negative XYL test with either the API 20C AUX or Vitek yeast identification system to provide a presumptive identification of C. dubliniensis. A negative MDG test result with either system would also be helpful but may misclassify C. albicans as C. dubliniensis, especially when the API 20C AUX system is used.

Candida↗

PubMed1999

In vitro susceptibilities of Candida dubliniensis isolates tested against the new triazole and echinocandin antifungal agents.

Candida dubliniensis is a newly recognized fungal pathogen causing mucosal disease in AIDS patients. Although preliminary studies indicate that most strains of C. dubliniensis are susceptible to established antifungal agents, fluconazole-resistant strains have been detected. Furthermore, fluconazole-resistant strains are easily derived in vitro, and these strains exhibit increased expression of multidrug resistance transporters, especially MDR1. Because of the potential for the development of resistant strains of C. dubliniensis, it is prudent to explore the in vitro activities of several of the newer triazole and echinocandin antifungals against isolates of C. dubliniensis. In this study we tested 71 isolates of C. dubliniensis against the triazoles BMS-207147, Sch 56592, and voriconazole and a representative of the echinocandin class of antifungal agents, MK-0991. We compared the activities of these agents with those of the established antifungal agents fluconazole, itraconazole, amphotericin B, and 5-fluorocytosine (5FC) by using National Committee for Clinical Laboratory Standards microdilution reference methods. Our findings indicate that the vast majority of clinical isolates of C. dubliniensis are highly susceptible to both new and established antifungal agents. Strains with decreased susceptibilities to fluconazole remained susceptible to the investigational agents as well as to amphotericin B and 5FC. The increased potencies of the new triazole and echinocandin antifungal agents may provide effective therapeutic options for the treatment of infections due to C. dubliniensis.

AIDS-Related Opportunistic Infections↗

PubMed1999

Rapid PCR test for discriminating between Candida albicans and Candida dubliniensis isolates using primers derived from the pH-regulated PHR1 and PHR2 genes of C. albicans.

The development of a satisfactory means to reliably distinguish between the two closely related species Candida albicans and Candida dubliniensis in the clinical mycology laboratory has proved difficult because these two species are phenotypically so similar. In this study, we have detected homologues of the pH-regulated C. albicans PHR1 and PHR2 genes in C. dubliniensis. Restriction fragment length polymorphism analysis suggests that there are significant sequence differences between the genes of the two species. In order to exploit this apparent difference, oligonucleotide primers based on the coding sequence of the C. albicans PHR1 structural gene were designed and used in PCR experiments. Use of these primers with C. albicans template DNA from 17 strains yielded a predicted 1.6-kb product, while C. dubliniensis template DNA from 19 strains yielded no product. We therefore propose that PCR using these primers is a rapid and reliable means of distinguishing the two germ tube- and chlamydospore-producing species C. albicans and C. dubliniensis.

Apoenzymes↗

PubMed1998

A common mechanism for blockade of heme polymerization by antimalarial quinolines.

The antimalarial quinolines are believed to work by blocking the polymerization of toxic heme released during hemoglobin proteolysis in intraerythrocytic Plasmodium falciparum. In the presence of free heme, chloroquine and quinidine associate with the heme polymer. We have proposed that this association of the quinoline-heme complex with polymer caps the growing heme polymer, preventing further sequestration of additional heme that then accumulates to levels that kill the parasite. In this work results of binding assays demonstrate that the association of quinoline-heme complex with heme polymer is specific, saturable, and high affinity and that diverse quinoline analogs can compete for binding. The relative quinoline binding affinity for heme polymer rather than free heme correlates with disruption of heme polymerization. Mefloquine, another important antimalarial quinoline, associated with polymer in a similar fashion, both in cultured parasites and in the test tube. In parasite culture, blocking heme release with protease inhibitor was antagonistic to mefloquine action, as it is to chloroquine action. These data suggest a common mechanism for quinoline antimalarial action dependent on drug interaction with both heme and heme polymer.

Animals↗

PubMed1998

Identification and characterization of an unusual double serine/threonine protein phosphatase 2C in the malaria parasite Plasmodium falciparum.

We have cloned a gene from Plasmodium falciparum with homology to the Mg2+-dependent serine/threonine protein phosphatase 2C (PP2C) family. The predicted coding region is 920 amino acids long, twice the size of other members of this family. We show that this protein can be divided into two halves (Pf2C-1 and Pf2C-2), each a complete phosphatase unit with homology to other phosphatases of this class. To study the function of this PP2C, we have tested the ability of different constructs to complement conditional null mutants of yeast. Our results show that expression of the full-length protein, the first half alone, the second half alone, or a hybrid with the N terminus of the first half and the C terminus of the second half was able to complement the heat shock response defect of a Schizosaccharomyces pombe strain with a PP2C (PTC1) deletion. Recombinant P. falciparum PP2C expressed in Escherichia coli was active in dephosphorylating 32P-labeled casein in an Mg2+- or Mn2+-dependent reaction. Each half alone was also active in recombinant form. Using the two-hybrid system, we have shown that the two halves can interact. Gel filtration assay of P. falciparum protein extracts suggests that full-length PfPP2C is a dimer, and phosphatase activity competition experiments indicate that dimerization of PfPP2C is required for its optimal activity. This unusual phosphatase molecule appears to be composed of four catalytic units on two polypeptide chains.

Amino Acid Sequence↗

PubMed1998

Distinct effects of allogeneic blood transfusion on splenocyte cytokine production after hemorrhagic shock.

UNLABELLED: Allogeneic blood transfusion is known to be immunosuppressive in the settings of cancer and transplantation, but the contribution of blood transfusion to immunomodulation after hemorrhage is unknown. Our purpose was to determine the influence of allogeneic blood transfusion upon cytokine profiles following hemorrhagic shock, using a model which approximates the clinical setting. METHODS: Male C3H/HeN mice were hemorrhaged via femoral arterial catheters to a mean arterial pressure (MAP) of 35 +/- 5 mm Hg, which was maintained for 1 h. Mice were resuscitated with autologous blood (auto BT) or allogeneic blood (allo BT) from Balb/c mice (both equivalent to volume of shed blood), and crystalloid (2X the volume of shed blood)-infused at 0.05 ml/min. Animals were sacrificed at 1, 2, and 5 days postshock, and splenocytes were cultured for 24 h with anti-CD3 antibody. Supernatants were assayed for IL-2, IL-6, IL-10, and gamma-IFN by ELISA. RESULTS: Regardless of transfusion status, hemorrhagic shock resulted in increased IL-6 and gamma-IFN by Day 2 postshock. Distinct cytokine alterations after allogeneic transfusion were as follows. IL-2: transient elevation of splenocyte IL-2 production in the shock + allo BT group (P < 0.005 vs. shock + auto BT) at Postshock Day 2. IL-6: suppression in IL-6 production in the shock + allo BT group by Postshock Day 5 (P < 0.05 vs. shock + auto BT). IL-10: persistently elevated IL-10 production following shock + allo BT (Day 1, P < 0.001 vs. shock + auto BT; Day 5; P < 0.05 vs. shock + auto BT). gamma-IFN: elevation in gamma-IFN production by Day 5 in the shock + allo BT group (P < 0.0005 vs. shock + auto BT). CONCLUSIONS: Allogeneic blood transfusion results in distinct alterations in splenocyte production of IL-2, IL-6, IL-10, and gamma-IFN after hemorrhagic shock. This model reflects the clinical usage of blood products and demonstrates some of the immune alterations after transfusion.

Animals↗

PubMed1998

Identification and expression of multidrug transporters responsible for fluconazole resistance in Candida dubliniensis.

Candida dubliniensis is a recently described Candida species associated with oral candidosis in human immunodeficiency virus (HIV)-infected and AIDS patients, from whom fluconazole-resistant clinical isolates have been previously recovered. Furthermore, derivatives exhibiting a stable fluconazole-resistant phenotype have been readily generated in vitro from fluconazole-susceptible isolates following exposure to the drug. In this study, fluconazole-resistant isolates accumulated up to 80% less [3H] fluconazole than susceptible isolates and also exhibited reduced susceptibility to the metabolic inhibitors 4-nitroquinoline-N-oxide and methotrexate. These findings suggested that C. dubliniensis may encode multidrug transporters similar to those encoded by the C. albicans MDR1, CDR1, and CDR2 genes (CaMDR1, CaCDR1, and CaCDR2, respectively). A C. dubliniensis homolog of CaMDR1, termed CdMDR1, was cloned; its nucleotide sequence was found to be 92% identical to the corresponding CaMDR1 sequence, while the predicted CdMDR1 protein was found to be 96% identical to the corresponding CaMDR1 protein. By PCR, C. dubliniensis was also found to encode homologs of CDR1 and CDR2, termed CdCDR1 and CdCDR2, respectively. Expression of CdMDR1 in a fluconazole-susceptible delta pdr5 null mutant of Saccharomyces cerevisiae conferred a fluconazole-resistant phenotype and resulted in a 75% decrease in accumulation of [3H]fluconazole. Northern analysis of fluconazole-susceptible and -resistant isolates of C. dubliniensis revealed that fluconazole resistance was associated with increased expression of CdMDR1 mRNA. In contrast, most studies showed that overexpression of CaCDR1 was associated with fluconazole resistance in C. albicans. Increased levels of the CdMdr1p protein were also detected in fluconazole-resistant isolates. Similar results were obtained with fluconazole-resistant derivatives of C. dubliniensis generated in vitro, some of which also exhibited increased levels of CdCDR1 mRNA and CdCdr1p protein. These results demonstrate that C. dubliniensis encodes multidrug transporters which mediate fluconazole resistance in clinical isolates and which can be rapidly mobilized, at least in vitro, on exposure to fluconazole.

ATP Binding Cassette Transporter, Subfamily B, Mem↗