NCCLS M27-A Research Articles

In vitro antifungal activity and phytochemical analysis of lichen Parmelia perlata (L.) Ach.

Fungal diseases like candidiasis and cryptococcosis are the commonest opportunistic infections. Since the difficulties associated with the management of antifungal infections, there is an urgent need for the development of new and effective antifungal agents. In this perspective, natural products are a viable alternative source. The lichen Parmelia perlata, commonly known as stone flower, is traditionally used in the Indian system of medicine. The aim of the present study was to evaluate in vitro antifungal activity of petroleum ether extract of Parmelia perlata against Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, and Cryptococcus neoformans and its phytochemical analysis. The anti-fungal activity of the extract of Parmelia perlata was determined by measuring the Minimal Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) by microbroth dilution method as per the CLSI (NCCLS M27-A2) guidelines. The extract of Parmelia perlata showed the MIC and MFC against Candida species between 600±28µg/ml to 690±16µg/ml and between 750±90µg/ml to 820±45µg/ml, respectively. In addition, the respective values against Cryptococcus neoformans were 550±17µg/ml and 690±34µg/ml, respectively. Further, Parmelia perlata has yielded an acid named- (+)-6-deacetyl-9b-carbmethoxy-9b-dimethylusnic acid and, a quinine named -6-acetyl-11-carbmethoxy-1,4,7,10-tetrahydroxy-2,8-dimethyl naphthacene-5,12-quinone, which were identified on the basis of spectroscopic studies as 2-acetyl-9b-carbmethoxy-7,9-dihydroxy-8-methyl-1,3(2H,9bH)-dibenzofurandione and naphthacene-5,12-quinone derivative, along with four well known compounds-tridecyl myristate, 3-ketooleanane, arachidyl alcohol and (+) usnic acid, respectively. The results of this study showed that Parmelia perlata is a promising antifungal agent.

In vitro evaluation of antifungal and cytotoxic activities as also the therapeutic safety of the oxidized form of amphotericin B

The aim of this study was to evaluate the antifungal and cytotoxic activities of the oxidized form of amphotericin B (AmB-Ox) as well as to determine whether oxidation process of AmB is therapeutically beneficial in vitro. The antifungal activity was estimated against Candida albicans ATCC 10231 and Candida parasilosis ATCC 22019 by broth microdilution method according to the NCCLS M27-A2 standards. The in vitro cytotoxicity was evaluated using normal green monkey kidney cells (GMK) by MTT assay. The obtained results demonstrated that AmB-Ox possesses 16-fold decreased antifungal properties against the two Candida strains and 5-fold lower cytotoxic activity towards GMK cells in comparison with AmB.The therapeutic safety in vitro assessed by calculating the ratio between cytotoxicity (CC50 value) to antifungal activity (MIC value) showed that oxidation of AmB is a very unfavourable process in vitro, because leads to formation of derivative (AmB-Ox) that lost antifungal properties much more rapidly than cytotoxic activity. Thus, the process of the oxidation of AmB in vivo (if it occurs) can be also highly harmful for patient.

Antifungal Activity of Oleoresin and Fractions of Pinus elliottii Engelm and Pinus tropicalis against Phytopathogens

Microorganism resistance to the existing products is yet another difficulty that agriculturalists have to deal with. In this context, the search for new agricultural products that can fight phytopathogens has become increasingly important. Plants have played an important role in this process, because they can serve as a source of new compounds for drug discovery. Plants belonging to the genus Pinus produce an oleoresin that protects the plant against herbivores and pathogens. With a view to developing products that can combat fungal pathogens without harming the environment, this work aimed to evaluate the antifungal activity of the oleoresins and fractions of Pinus elliottii Engelm and Pinus tropicalis against phytopathogens. The methodology based on NCCLS M38-A standards aided antifungal activity assessment. The microdilution method helped to determine the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC). The oleoresins of P. elliottii and P. tropicalis afforded the most significant results—they displayed fungicidal activity against all the tested species. MIC values were promising, especially the MIC of the oleoresin of P. elliottii against S. rolfsii (1.95 μg·mL-1). The MIC values of the oleoresins of P. elliottii and P. tropicalis ranged from 1.95 to 1000 μg·mL-1 and from 31.25 to 250 μg·mL-1, respectively. Fraction PT2 of P. tropicalis furnished the best results among all the assayed fractions: MIC values lay between 125 and 500 μg·mL-11. In conclusion, the oleoresin of P. tropicalis is a promising source of new antifungal agents for application in the treatment of phytopathogenic infections.

Grey relational analysis on fingerprint characteristics of different eluted parts of Euphorbia humifusa and antifungal effect

To determine the contribution of chemical components represented by characteristic peaks of fingerprint of different eluted parts of Euphorbia humifusa to the antifungal effect to provide theoretical basis and data support for the traditional Chinese medicine quality control mode. HPLC was introduced to analyze fingerprint of different eluted parts of E. humifusa. The antifungal effect was observed using NCCLS M38-A method. The correlation between fingerprint and antifungal effect of E. humifusa was identified using grey relational analysis. The antifungal effect of different eluted parts of E. humifusa is resulted from the combined action of multiple chemical components. Chemical components representing a contribution to pharmacy dynamics were ordered by representative spectrum 2 > 5 > 3 > 4 > 1 for anti-Trichophyton rubrum and anti-T. mentagrophytes 5 > 2 > 3 > 4 > 1. Fingerprint of different eluted parts of E. humifusa is related with their antifungal effect to some extent.

Atividade inibitória do óleo essencial de orégano em fungos de importância médica e veterinária

ABSTRACT The in vitro activity of Origanum vulgare essential oil against fungal isolates was evaluated. A total of 27 clinical isolates were used, including: C. albicans, S. schenckii, M. pachydermatis , Aspergillus flavus , and A. fumigatus. Microdilution in broth technique (NCCLS M27-A2 and M-38) was used and susceptibility was expressed as Minimum Inhibitory Concentration (MIC). Essential oil was obtained by hydrodistillation in Clevenger and analyzed by gas chromatography, showing the presence of 4-terpineol, alpha-terpineol, 4-terpinene, thymol and carvacrol, as the main compounds. Origanum oil MIC for C. albicans varied from 125 to 50 0μ L/mL; for S. schenckii, from 250 to 500μ L/mL; for M. pachydermatis, from 15 to 30μ L/mL; and for Aspergillus, from 30 to 60  L/mL. Isolates sensitivity showed to the origanum oil stimulates the accomplishment of new studies, including in vivo tests, contributing to the search of alternative treatments to mycosis. Keywords: fungal, essential oil, origanum

In Vitro Activity of Amphotericin B Against Candida lusitaniae Clinical Isolates

" In Vitro Activity of Amphotericin B Against Candida lusitaniae Clinical Isolates." Journal of Chemotherapy, 22(1), pp. 71–72

Enteropatógenos relacionados à diarréia em pacientes HIV que fazem uso de terapia anti-retroviral

The etiology of the diarrheic process in AIDS may be caused by viruses, bacteria, fungi, protozoa or helminths, as well as HIV itself. This study evaluated enteropathogens relating to diarrhea in HIV patients who were on antiretroviral therapy. The parasitological methods used were Faust, Hoffmann and Kinyoun. Isolation and culturing of fungi were carried out in accordance with the methodology recommended by the NCCLS M27-A standard. The yeast species were identified using the polymerase chain reaction (PCR). Bacteria were isolated on MacConkey and SS agar and the species were identified using Enterokit B (Probac do Brasil) and biochemical methods. Forty-nine patients were evaluated: 44.89% presented enteroparasites and 48.1% presented Candida sp, of which 61.5% were Candida albicans, 7.6% were Candida sp and 30.7% were Candida non-albicans. Bacteria were isolated from 72% of the patients, of which 49% were Escherichia coli, 13% Salmonella parathyphi, Klebsiella sp or Proteus and 6% Citrobacter freundii or Yersinia sp. There was high prevalence of Candida sp in HIV patients with diarrhea and non-albicans species were isolated. Their presence could be taken to mean that they were accomplices in or causes of the infection.

Myricetin from Plinia cauliflora (Mart.) Kausel and activity against Candida albicans

The aim of this work was to obtain a pure compound from P. cauliflora leaves extract and to evaluate its anti-Candida activity. The leaves extract was obtained with 70% ethanol by percolation. After partition with ethyl acetate and water, the ethyl acetate fraction was chromatographed through a Sephadex LH-20 gel column eluted with methanol. Fractions (8 mL each) were collected and analyzed by TLC (silicagel plate, 025µm thickness) eluted with chloroform:methanol:n-propanol:water (5:6:1:4; v/v; organic phase) and sprayed with sulphuric anisaldehyde. Spots with similar Rf and colour were assembled The isolated compound was identified by its spectral data (1H-NMR, 13C-NMR, HMQC, COSY, HMBC, 1D-TOCSY) and compared to those reported in the literature [1,2]. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) was performed according NCCLS M27-A2 [3]. The identified fraction sample was diluted to 500µg/mL and the Candida albicans (ATCC 64548) final inoculum was 2.5–5.0×103 cells/mL. The spectroscopic data of the isolated compound were in complete agreement with the flavonoid myricetin-3-O-β-allopyranoside. This compound showed MIC and MFC of 250µg/mL and 500µg/mL, respectively. This was the first flavonol isolated from the leaves of P. cauliflora and it was possible to determine for the first time its good activity against the pathogen C. albicans.

Caspofungin Use for the Treatment of Invasive Aspergillosis–Results of a Prospective Observational Registry in the Asia Pacific Region

resistance as exemplified by fluconazole and echinocandinsresistant Candida spp. This situation highlights the need for advent of safe, novel and effective antifungal compounds. The objective of this study was to determine whether secondary metabolites produced by a species of Streptomyces was able to exert antifungal effects against seven and five medically important yeast Candida spp. and molds Aspergilllus spp. respectively. NCCLS M27A and M38A methods for MIC determination were followed stringently in this screening procedure. Our initial work found all of these fungi were susceptible to the crude fermentative acetone extract (MIC ≤0.625mg/ml for Candida spp. and ≤2.5mg/ml for Aspergillus spp.). Five fluconazole-resistant clinical strains were also sensitive to it, with MIC values ≤0.312mg/ml. In addition, it also totally inhibited C. albicans ATCC 14053 hyphae growth at 0.5mg/ml. These promising results showed that potential antifungal compounds from secondary metabolites were produced by this species of Streptomyces. This work will be further tested with fractionated extracts and the mechanism of action of potential bioactive compounds will be elucidated.

Sinergismo entre ajoeno y ketoconazol en aislamientos de Microsporum canis. Un estudio preliminar mediante la determinación de la concentración inhibitoria fraccional (CIF)

Fungal infections are probably the most frequent infectious diseases affecting human being. Resistance to different anti-fungal drugs, and their bioavailability in the infection site, represent a problem for treatment. Looking for effective solutions, combination of two or more antifungal drugs to obtain an additive effect or synergic effect that potent antifungal activity has been investigated. In this study, the effect (additive, antagonist or synergistic) of ajoene and ketoconazole combination was evaluated in the growth and proliferation of filamentous fungi. Interactions in vitro were investigated in three isolates of Microsporum canis through a preliminary study using micro dilution, according to recommendations of NCCLS M-38A, with several modifications. Results obtained for CIF of each isolates studied (CIF = 0.18 0.36 microM), demonstrate that exists a very potent synergistic effect, when they are combined, and it represents a hope for future clinic trials to treat resilient fungal infections caused by M. canis.

In vitro evaluation of terbinafine and itraconazole against dematiaceous fungi

Two hundred and three isolates representing 15 species of filamentous ascomycetes were evaluated against terbinafine and itraconazole using a modification of the NCCLS M27-A standard reference method for yeasts. The MIC ranges and geometric means were similar, although terbinafine tended to have the lowest values. The loculoascomycete clade tested had consistently low MIC geometric mean values for its members, ranging from 0·03 to 0·17 μg ml−1 for terbinafine and 0·03–0·37 μg ml−1 for itraconazole.

Susceptibility profile of yeast-like organisms isolated from HIV/AIDS patients; using NCCLs macrodiltion method compared with agar diffusion technique.

Yeast like opportunistic fungal infection has been reported globally amongst HIV/AIDS patients, particularly as the etiologic agent of oral thrush. Fluconazole antibiotic has been most popularly employed in treating cases of oral thrush in HIV/AIDS patients. Recent reports have recorded antifungal drug resistance amongst immunocompromised subjects. This constitutes a big problem in the management of opportunistic candidiasis. The NCCLS micro/macrodilusion sensitivity testing procedure is expensive, cumbersome and requires a level of sophistication. This study was designed to compare NCCLS M-27-A macrodilution method (expensive) with agar diffusion technique (cheap and simple), to provide a reliable rapid alternative to the new pressing need for antifungal routine sensitivity testing. Sputum specimens from 213(108 females and 105 males) HIV positive patients were plated onto SDA. The isolates were identified by morphotyping, microscopy and speciated using germ tube test, and battery of biochemical sugar fermentation; and assimilation tests. Fluconazole agar diffusion susceptibility testing was carried out on each isolate, compared with the NCCLS macrodilution sensitivity assay standard. Of the 74 isolates tested for fluconazole sensitivity, 59(79.7%) were sensitive (zone diameter > 19mm, mean diameter 28mm), 6(8.1%) were Sensitive Dose Dependent (S-DD) (zone diameter 13-18mm, mean diameter 16mm), while 9(12.2%) were resistant (zone diameter 64μg/ml profile, using the NCCLS macrodilution assay. The differences between the test method (Agar diffusion) and the control standard method (NCCLS-M 27-A broth Macrodilution MICS) were not statistically significant using t-test (two tail) (t = 4.302656, P=1.0). Among the C. albicans isolates, 26(86.7%) were sensitive to fluconazole. The rank of susceptibility was C. albicans > C. tropicalis > C. krusei. It is concluded that broncho-oro-pharyngeal Candida and other yeast-like species existed in about one third of the HIV and AIDS patients studied; in which C. albicans was the most prevalent, while about ten percent of all the Candida isolates were resistant to fluconazole. The reliability of germ tube production as a confirmatory test for Candida albicans in HIV infection was as high as 96.7% and is therefore, recommended for continued use. Agar diffusion compared favourably with the NCCLS macrodilution technique, hence it is recommended for routine antifungal sensitivity test on all isolates of yeast-like cells from HIV and AIDS subjects. Keywords : HIV/AIDS, oral thrush, yeast-like cells, fluconazole resistance, NCCLS vs agar diffusion technique. African Journal of Clinical and Experimental Microbiology Vol. 9 (2) 2008 pp. 88-96

Atividade in vitro do óleo essencial de Origanum vulgare frente à Sporothrix Schenckii

In vitro activity of the essential oil Origanum vulgare against Sporothrix schenckii was determined by the MIC (Minimum Inhibitory Concentration). For this, seven samples of S. schenckii were studied, two isolated from two cases of human sporotrichosis and five isolated from cats. Analysis of the essential oil was carried out in a gas chromatograph (GC/FID) for the identification and quantification of thymol and carvacrol (antifungal agents). MIC was obtained based on the microdilution method according to the adapted document NCCLS-M 27A2 for fitopharmacy. All the isolates presented sensibility to the essential oil. S. schenckii was inhibited in a concentration of 0.25% (250m l/ml). Chromatographic analysis showed that thymol concentration was bigger than carvacrol. The antifungal activity demonstrated by the essential oil of O. vulgare against S. Schenckii stimulates the accomplishment of more studies, including in vivo studies.

Potent anti‐microbial activity of traditional Chinese medicine herbs against Candida species

Anti-candidial activities of eight traditional Chinese medicinal (TCM) herbs were evaluated against six different Candida species. TCM preparations were screened for antifungal activity using a standard agar diffusion assay. Following identification of potential candidate herbs, their minimum inhibitory concentration (MIC) values were determined using the standardised NCCLS M-27A broth microdilution assay. Among TCM herbs, Rhizoma Coptidis had potent antifungal activity against Candida glabrata, Candida krusei and Candida tropicalis, but not against Candida albicans, Candida dubliniensis and Candida parapsolosis. The MIC values of the Rhizoma Coptidis against C. glabrata, C. krusei and C. tropicalis were 50, 50 and 100 microg ml(-1) respectively. We report here, for the first time, the potent antifungal activity of Rhizoma Coptidis and Cortex phellodendri Chinesis on three different non-albicans Candida species, C. glabrata, C. krusei and C. tropicalis and hence their possible use as therapeutic agents.

In Vitro Susceptibility of Sporothrix schenckii to Six Antifungal Agents Determined Using Three Different Methods

The in vitro susceptibility of Sporothrix schenckii to antifungal drugs has been determined with three different methods. Nineteen Peruvian clinical isolates of S. schenckii were tested against amphotericin B (AB), flucytosine (FC), fluconazole (FZ), itraconazole (IZ), voriconazole (VZ), and ketoconazole (KZ). Modified NCCLS M38-A, Sensititre YeastOne (SYO), and ATB Fungus 2 (ATBF2) methods were used to determine the MICs. ATCC isolates of Candida parapsilosis, Candida krusei, and Aspergillus flavus were used for quality control. Sporothrix inocula were prepared with the mycelial form growing on potato dextrose agar at 28 +/- 2 degrees C. MICs of AB, FC, FZ, and IZ were determined with all three methods, VZ with M38-A and SYO, and KZ with only SYO. The three methods showed high MICs of FZ and FC (MIC(90) of 0.5 microg/ml), being homogeneously lower than those of IZ and KZ. The M38-A method showed a variable MIC range of VZ (4.0 to 16 microg/ml); the geometric mean (GM) was 9.3 mug/ml. The MIC range of AB was wide (0.06 to 16 microg/ml), but the GM was 1.2 microg/ml, suggesting that the MIC is strain dependent. Agreement (two log(2) dilutions) between commercial techniques and the modified M38-A method was very high with FZ, IZ, and FC. In AB and VZ, the agreement was lower, being related to the antifungal concentrations of each method. The highest activity against S. schenckii was found with IZ and KZ. Lack of activity was observed with FZ, VZ, and FC. When AB is indicated for sporotrichosis, the susceptibility of the strain must be analyzed. Commercial quantitative antifungal methods have a limited usefulness in S. schenckii.

Correlation between the E test and the CLSI M-38 A microdilution method to determine the activity of amphotericin B, voriconazole, and itraconazole against clinical isolates of Aspergillus fumigatus

The in vitro activities of amphotericin B, itraconazole, and voriconazole against 283 clinical isolates of Aspergillus fumigatus were studied by comparing the E test method with the reference procedure NCCLS (CLSI) M-38A. The methods were considered to agree when the results of the MICs by E test were within ±2 dilutions of the MICs obtained by CLSI M-38 A. Agreement of readings at 24, 48, and 72 h of incubation was as follows: amphotericin B: 4.2%, 98%, and 40.6%; itraconazole: 92.5%, 100%, and 89.9%; and voriconazole: 98.9%, 100%, and 99.7%. The correlation between methods to classify the strains as “susceptible” or “resistant” was very good for itraconazole and voriconazole after 48 h of incubation, but 23% of the strains were incorrectly classified by E test for amphotericin B. The E test is an efficacious method for antifungal susceptibility testing in A. fumigatus for itraconazole and voriconazole when the plates are read after 48 h of incubation. The use of the E test to study the activity of amphotericin B should be avoided.

In vitro activity and synergism of amphotericin B, azoles and cationic antimicrobials against the emerging pathogen Trichoderma spp.

The uncommon fungal pathogen Trichoderma shows increasing medical importance particularly in immunocompromised patients. Despite systemic antifungal therapy, prognosis of Trichoderma infection is poor regardless of the type of infection and the therapy used. The aim of the present study was to evaluate the in vitro activity and synergism of double antifungal combinations including amphotericin B, voriconazole, fluconazole, chlorhexidine digluconate and Akacid plus against 15 isolates of Trichoderma longibrachiatum and 1 isolate of Trichoderma harzianum. Individual MICs were determined by using broth microdilution method following the NCCLS M38-A guidelines with standard RPMI 1640 broth. Synergy tests were performed using the chequerboard method. All clinical Trichoderma strains showed reduced susceptibility to fluconazole (MICs>or=64 mg/L) and amphotericin B (MICs=2 mg/L), whereas lower MICs of 0.5-1 mg/L were detected for voriconazole. Akacid plus reached the lowest MIC values in a range of 0.06-0.5 mg/L, 4- to 32-fold higher MICs were found for chlorhexidine. No antagonism was observed for any of the antifungal combinations tested. Interaction of amphotericin B and azoles was indifferent (fractional inhibitory concentration index, FICI 2-4). The combination of one azole and one cationic biocide showed different degree of synergism (FICI 0.07-2.03). Interaction of Akacid plus and chlorhexidine resulted in synergism for each Trichoderma isolate (FICI-range 0.05-0.5). These results demonstrate no interaction between antifungals and some degree of synergism between azoles and cationic antimicrobials against Trichoderma spp.

Comparison of Sensititre YeastOne® with the NCCLS M38-A microdilution method to determine the activity of amphotericin B, voriconazole, and itraconazole against clinical isolates of Aspergillus fumigatus

The in vitro activities of amphotericin B, itraconazole, and voriconazole against 279 clinical isolates of Aspergillus fumigatus were studied by comparing Sensititre YeastOne® with the reference method NCCLS (CLSI) M38-A. The methods were considered to agree when the results of the MICs by Sensititre were within 2 dilutions of the MICs obtained by NCCLS M38-A. Agreement of readings at 24, 48, and 72 h was as follows: amphotericin B (0%, 31.5%, 82.1%), itraconazole (86.7%, 98.3%, 98.2%), and voriconazole (92.8%, 98.3%, 98.9%). Sensititre YeastOne® is a suitable alternative for the determination of the MICs of itraconazole and voriconazole against A. fumigatus when results are read at 48 h of incubation.

In vitro anticandidal activity of xanthorrhizol isolated from Curcuma xanthorrhiza Roxb

Xanthorrhizol, isolated from the methanol extract of Curcuma xanthorrhiza Roxb., was investigated for its anticandidal activity using six Candida species. The in vitro susceptibility tests for xanthorrhizol were carried out in terms of MIC and minimal fungicidal concentration (MFC) using the NCCLS M27-A2 broth microdilution method. Time-kill curves were determined to assess the correlation between MIC and fungicidal activity of xanthorrhizol at concentrations ranging from 0 MIC to 4x MIC. All Candida species showed susceptibility to xanthorrhizol in the MIC range 1.0-15.0 mg/L for Candida albicans, 1.0-10 mg/L for Candida glabrata, 2.0-8.0 mg/L for Candida guilliermondii, 2.5-7.5 mg/L for Candida krusei, 2.5-25 mg/L for Candida parapsilosis and 2.0-8.0 mg/L for Candida tropicalis. Time-kill curves demonstrated that xanthorrhizol was able to kill the Candida strains with MFCs of 20 mg/mL, 15 mg/mL, 12.5 mg/mL, 10 mg/L, 30 mg/mL and 10 mg/L for C. albicans, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis and C. tropicalis, respectively. The potent anticandidal activity of xanthorrhizol may support the use of C. xanthorriza for the treatment of candidiasis.

Fluconazole resistance mechanisms inCandida krusei: The contribution of efflux-pumps

The main resistance mechanism for fluconazole in Candida krusei is the diminished sensitivity of the target enzyme cytochrome P450 sterol 14 alpha-demethylase (CYP51) to inhibition by azole agents. An alternative mechanism of resistance, efflux-pump activity, has been proposed. The aim of our study was to find out the possible contribution of efflux-pumps in conferring resistance to fluconazole in 33 C. krusei isolates from different clinical sources. The activity of efflux-pumps was checked using the inhibitor CCCP (carbonyl cyanide 3-chloro-phenylhydrazone), which decreases the minimum inhibitory concentration (MIC) when resistance is attributed. We established a concentration of 0.5 microg/ml of CCCP. The susceptibility patterns of our isolates for five antifungal drugs (amphotericin B, fluconazole, itraconazole, flucytosine and voriconazole) were determined according to an NCCLS M27-A2 protocol modification (Sensititre Yeast One). We tested all the strains before and after adding CCCP to the RPMI medium. The MIC90s and ranges of the drugs were identical before and after addition of CCCP. The MIC for fluconazole was higher than for the other antifungals. The new triazoles were active and the MICs were lower, although this should be interpreted carefully as the drugs showed different cut-offs. Only one isolate showed a two-fold decrease in MIC to fluconazole when CCCP was added. We did not find any multi-resistant strains. According to our study with C. krusei, CCCP-inhibited efflux-pumps do not play a significant role in resistance to fluconazole.