1 Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia Medica, 2 Facoltà di Farmacia, and 3 Dipartimento Farmaco Chimico Tecnologico, Università di Cagliari, Cagliari, Italy; 4 Ospedale SS. Trinità, Cagliari, Italy
Received 19 October 2004; returned 10 December 2004; revised 28 January 2005; accepted 2 February 2005
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Abstract |
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Methods: We investigated the activity of 2-cyclohexylidenhydrazo-4-phenyl-thiazole (EM-01D2) against 114 clinical isolates of Candida spp., representing five different species, by microdilution, according to the NCCLS method 27-A. The activity against C. albicans biofilms was also investigated. Toxicity in vitro was evaluated by MTT reduction assay.
Results: EM-01D2 demonstrated low toxicity, broad spectrum, fungicidal activity and was active against C. albicans and Candida krusei at concentrations lower than those shown by amphotericin B and fluconazole (P < 0.05). It maintained potent in vitro activity against fluconazole-resistant C. albicans isolates. Fungicidal activity occurred at concentrations 12 doubling dilutions greater than the corresponding MICs, and timekill analysis indicated that a 99.9% loss of C. albicans viability occurred after 6 h of incubation in the presence of EM-01D2 at concentrations equal to four times the MIC. EM-01D2 was also active in inhibiting the growth of C. albicans ATCC 10231 biofilms, even though such inhibition occurred at concentrations higher than the MICs determined under planktonic growth conditions. However, when C. albicans biofilms were pre-exposed to subinhibitory concentrations of EM-01D2, a reduction of MIC50 of amphotericin B was observed.
Conclusions: Based on these results, EM-01D2 could represent a template for the development of novel fungicidal agents.
Keywords: antifungal agents , biofilms , isothiosemicarbazone derivatives
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Introduction |
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The use of azole prophylaxis, in particular fluconazole and itraconazole, to prevent fungal infections is the standard procedure in many institutions. However, it has been demonstrated that cells of C. albicans that are pre-exposed to azoles are protected from subsequent killing by exposure to amphotericin B.3
Recently it has been demonstrated that ergosterol biosynthesis inhibitors become fungicidal against C. albicans and other Candida species, such as Candida krusei and Candida glabrata, which are known for intrinsic or rapidly acquired resistance to azoles, when combined with calcineurin inhibitors such as ciclosporin A or tacrolimus.4 However, ciclosporin A is well known for its toxicity to renal tubular cells due to increased intracellular calcium concentration and thus to a stimulation of the activity of calcium-dependent calpains and caspases.5,6 Therefore, the use of the combination ergosterol biosynthesis inhibitors and calcineurin inhibitors has still to be carefully evaluated and standardized, and underlines the need for new fungicidal drugs with low toxicity for therapy of fungal infections.
New semisynthetic echinocandins, such as caspofungin and micafungin, are fungicidal water-soluble molecules that inhibit synthesis of 1,3-ß-D-glucan, a main structural component of the fungal cell wall.1,7 They display excellent toxicological profiles and are usually active against several clinically important fungi, in particular Candida spp. and Aspergillus spp.8,9 They have also proven to be active against isolates of C. albicans with high levels of fluconazole resistance. Furthermore, the activities of caspofungin and lipid formulations of amphotericin B against C. albicans and Candida parapsilosis biofilms are comparable.10,11 This aspect is particularly noteworthy since biofilms represent the most prevalent type of microbial growth in nature, are crucial to the development of clinical infections and their susceptibility to antifungal agents is usually dramatically reduced. However, the therapy of fungal infections with echinocandins is still expensive and often not affordable, in particular in developing countries. Because of the importance of fungal infections, in particular in compromised patients, the limitations of currently available antifungal agents regarding their toxicities, and the increasing prevalence of pathogen resistance, new fungicidal agents are needed.
The antimicrobial activity of several isothiosemicarbazones and their cyclic analogues have been recently described, and encouraging results have been obtained with some structurally related compounds, in particular against Candida spp.12 In this report, we describe the evaluation of the toxicity and in vitro activity of 2-cyclohexylidenhydrazo-4-phenyl-thiazole (EM-01D2), a cyclic analogue of isothiosemicarbazone, against 114 clinical isolates of Candida spp., representing five different species and 28 isolates of fluconazole-resistant C. albicans, in comparison with amphotericin B and fluconazole.
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Materials and methods |
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A total of 114 strains of Candida spp. recently isolated in our laboratories were selected for this study. The isolates included were C. albicans (52 strains), C. krusei (23), C. parapsilosis (17), C. glabrata (11) and Candida tropicalis (11). The majority of isolates were obtained from blood or normally sterile body fluids. They were identified by standard methods. Quality control strains C. parapsilosis ATCC 22019, C. krusei ATCC 6258 and C. albicans ATCC 90028 were included in each test, as recommended by the NCCLS.13 Prior to testing, each isolate was subcultured at least twice on potato dextrose agar plates (Difco, BD Biosciences, Bedford, MA, USA) to ensure purity and optimal growth.
Antifungal agents
EM-01D2 (Figure 1) belongs to a series of isothiosemicarbazone cyclic analogues previously described.12
It was obtained by the reaction of equimolar amounts of cycloalkylthiosemicarbazone and -halogen ketone under reflux in isopropyl alcohol until complete dissolution. When the foaming product was formed, the mixture was allowed to cool and the solid filtered. The product was crystallized from water/ethanol. Amphotericin B was purchased from Sigma Chemicals Co. (St Louis, MO, USA). Fluconazole was obtained from a commercially available iv formulation (Diflucan®, Pfizer Italia S.p.A.) at 200 mg/100 mL in saline. EM-01D2 and amphotericin B were dissolved in dimethyl sulphoxide at 10 mg/mL, for antifungal susceptibility studies, or 100 mg/mL, for cytotoxicity assays, and stored at 20°C until use.
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Vero cell lines used in this study were obtained from the Istituto Zooprofilattico della Lombardia e dell'Emilia (Brescia, Italy). Cells were grown and maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine, penicillin 50 IU/mL, streptomycin 50 mg/L and 10% fetal bovine serum (Gibco, Invitrogen, Carlsbad, CA, USA). Cultures were maintained at 37°C in 5% CO2. For cytotoxicity experiments, cells were seeded in 96-well plastic microtitre plates (Falcon, BD Biosciences) at a density of 1 x 104 cells/well in RPMI 1640 containing no antibiotics and EM-01D2, fluconazole or amphotericin B at concentrations in the range 0.191000 mg/L and incubated at 37°C in 5% CO2. Cells were observed for morphological changes at 24, 48 and 72 h of incubation. After 72 h the effects on the proliferation of Vero cells were determined by tetrazolium-based colorimetric MTT assay.14 The 50% cell-inhibitory concentration (IC50) reduced by 50% the optical density values (OD540, 690) with respect to control no drug-treated cells.
Antifungal susceptibility studies
MIC assays. MICs were determined by the broth microdilution method according to the NCCLS reference document M27-A.13 RPMI 1640 medium (Sigma Chemicals) without sodium bicarbonate, supplemented with L-glutamine (Gibco, Invitrogen) and buffered with 0.165 M MOPS (Sigma Chemicals) at pH 7.0 was used as test medium. Two-fold dilutions of the drugs with concentrations in the range 0.008200 mg/L were obtained in RPMI 1640 and dispensed into the wells of plastic microdilution trays. Starting inoculum suspensions were obtained by the spectrophotometric method of inoculum preparation, adjusted to 106 cfu/mL and then diluted in test medium to 2 x 104 cells/mL. A 100 µL yeast inoculum was added to each well of the microdilution trays to obtain final concentrations of the drugs ranging between 0.004100 mg/L and final inocula of 104 cells/mL. The inoculated plates were incubated overnight at 35°C in a humid atmosphere. After agitation, plates were read visually with the aid of a reading mirror and spectrophotometrically with an automatic plate reader (Sunrise Tecan, Grödig/Salzburg, Austria) set at 450 nm. For EM-01D2 and amphotericin B, MICs were determined at the lowest concentration at which a 100% inhibition of growth compared with drug-free control wells was observed. The MICs of fluconazole were read as the lowest concentration of drug that inhibits growth by 80%. MICs determined either visually or by spectrophotometric evaluation showed excellent agreement.
Minimum fungicidal concentration assays. After the MIC determination, a 100 µL sample from each well was seeded on plates of Sabouraud dextrose agar. Plates were incubated for 72 h at 35°C. The minimum fungicidal concentration (MFC) was defined as the minimum concentration of compound that resulted in the growth of less than two colonies representing the killing of > 99% of the original inoculum.
Timekill analysis. C. albicans isolate 1504 was grown overnight at 35°C on Sabouraud dextrose agar. Isolated colonies were selected and suspended in 0.9% NaCl to a turbidity equivalent to that of a 0.5 McFarland Standard. Flasks containing RPMI 1640 buffered with MOPS 0.165M to pH 7.0 plus EM-01D2 or amphotericin B at 4 x MIC (3.12 mg/L) or no antibiotic (growth control) were inoculated with the yeast suspension to a final concentration of 105 cfu/mL. The cultures were incubated at 35°C for up to 24 h. At the indicated times, aliquots were removed and the numbers of viable cells per millilitre determined on Sabouraud dextrose agar after serial dilutions in saline.
Antifungal susceptibility testing of C. albicans biofilms
C. albicans isolates 1298, 1411 and ATCC 10231 biofilms were formed in polystyrene, flat-bottomed, 96-well plates as previously described.15 Cells were propagated on yeast peptone dextrose (YPD) agar [1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose (Sigma Chemicals)], inoculated in YPD broth and incubated overnight at 30°C. Under this condition C. albicans grew in the budding yeast phase. Cells were harvested, washed in sterile PBS (10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride, pH 7.4) and resuspended in RPMI 1640 supplemented with L-glutamine and buffered with MOPS (pH 7.0) to a cellular density of 1 x 106 cells/mL. 100 µL of cell suspension was transferred into selected wells of flat-bottomed 96-well microtitre plates and incubated at 37°C for 48 h. After biofilm formation, the medium was discarded and non-adherent cells were removed by thoroughly washing three times in sterile PBS. Amphotericin B, fluconazole and EM-01D2 were then added to wells at serial doubling dilutions in RPMI 1640, and the biofilms were incubated in the presence of antifungal agents at final concentrations in the range 1000.004 mg/L at 37°C for 48 h. Untreated biofilms containing RPMI 1640 and no antifungal agent were included as negative control. Quantification of C. albicans biofilms was performed by the 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide (MTT; Sigma Chemicals) reduction assay.16,17 MTT was prepared as saturated solution at 500 mg/L in Ringer's lactate, filter sterilized through a 0.22 µm pore-size filter, aliquoted and stored at 70°C. An aliquot of the stock solution of MTT was thawed prior to each assay and menadione (Sigma Chemicals) 20 mM in acetone was added to obtain a final concentration of 1 µM. A 100 µL aliquot of the MTTmenadione solution was added to each well and plates were incubated for 2 h at 37°C. The colorimetric change produced by the MTT reduction was measured in a microtitre plate reader (Sunrise Tecan) at 490 nm. The antifungal concentration, which caused a 50% reduction in metabolic activity (50% RMA) of biofilms compared with control, was then determined. In this assay, the 50% RMA is equivalent to the MIC50 (minimum drug concentration at which 50% growth inhibition compared with untreated control is observed) as determined by the NCCLS M27-A method.18
Alteration of in vitro susceptibility of C. albicans biofilms to amphotericin B by pre-exposure to EM-01D2. The effect of pre-exposure of EM-01D2 on the activity of amphotericin B against C. albicans ATCC 10231 biofilms was also evaluated. Biofilms formed as described above were incubated at 37°C for 24 h in the presence of EM-01D2 10 mg/L. Under such conditions, no significant reduction in the metabolic activity of C. albicans was determined by the XTT reduction assay. Pre-exposed C. albicans biofilms were then incubated for 48 h with amphotericin B in RPMI 1640 at concentrations in the range 1000.04 mg/L and processed as described for the XTT reduction assay.
Data analysis
The MIC50 and MIC90 values were calculated as the concentrations of antifungal agents that were able to inhibit the growth of 50 and 90% of the isolates, respectively. The MFC50 and MFC90 values were calculated as the minimum fungicidal concentrations, as determined against 50 and 90% of isolates, respectively. Geometric mean MICs and MFCs were obtained to facilitate comparisons of the activities of the three drugs. Statistical analysis was performed by the unpaired two-tailed Student's t-test, and differences with P values of < 0.05 were considered significant.
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Results |
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No reduction of the metabolic activity of Vero cells was observed by the MTT reduction assay even at the highest concentration of EM-01D2 tested (1000 mg/L). The IC50s determined for amphotericin B (24.4 mg/L) and for fluconazole ( > 1000 mg/L) are consistent with data previously reported by others.
In vitro activity of EM-01D2
The in vitro activity of EM-01D2 was assessed against 114 isolates of yeasts. MICs and MFCs are reported in Tables 1 and 2, respectively. The activity of EM-01D2 was significantly superior (P < 0.05) to those of amphotericin B and fluconazole against fluconazole-susceptible C. albicans (MIC50 0.04, 0.39 and 0.78 mg/L, for EM-01D2, amphotericin B and fluconazole, respectively) and against C. krusei (MIC50 0.09, 0.78 and 50 mg/L, for EM-01D2, amphotericin B and fluconazole, respectively) isolates (Table 1). EM-01D2 was also highly active against fluconazole-resistant C. albicans isolates and its activity was comparable with that shown by amphotericin B. The MIC at which the 50% of strains were inhibited (MIC50), as determined by the broth microdilution method, was 0.39 mg/L for both EM-01D2 and amphotericin B.
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MFC assays
EM-01D2 was shown to be fungicidal against all the strains tested at concentrations 12 doubling dilutions greater than the corresponding MICs (Table 2). MFC50s determined for EM-01D2 against fluconazole-susceptible C. albicans (0.19 mg/L) and C. krusei (0.39 mg/L) were lower than those determined for amphotericin B (0.78 and 1.56 mg/L for C. albicans and C. krusei, respectively). EM-01D2 was also fungicidal against fluconazole-resistant C. albicans, with MFCs in the range 0.093.12 mg/L, and against C. parapsilosis, C. glabrata and C. tropicalis isolates.
Timekill analysis
Against the clinical isolate C. albicans 1504, the MICs obtained were 0.78 mg/L for both EM-01D2 and amphotericin B. When the two compounds were tested at 4 x MIC, EM-01D2 caused a 90.599.9% loss of yeast viability after 2 and 6 h of incubation, respectively, whereas amphotericin B caused a 92.6 and a > 99.9 % loss of viability after 1 and 4 h of incubation, respectively (Figure 2).
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The MTT reduction assay showed a reduction of the metabolic activity of cells when pre-formed C. albicans ATCC 10231 biofilms were exposed to EM-01D2 (Figure 3). However, the 48 h MIC50 of EM-01D2 for sessile C. albicans cells within biofilms was 29.20 mg/L, significantly higher than the MICs determined under planktonic growth conditions. The comparison with the antifungal agents used as reference drugs showed that fluconazole had no effects and no RMA was observed even at 100 mg/L, whereas amphotericin B showed an MIC50 at 0.97 mg/L. Such results are consistent with previously reported data.15 More interestingly, when C. albicans biofilms were pre-exposed to subinhibitory concentrations of EM-01D2 for 24 h, a reduction of MIC50 of amphotericin B to 0.07 mg/L was observed. No significant differences were detected in experiments carried out employing biofilms obtained from C. albicans clinical isolates 1298 and 1411.
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Discussion |
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Many Candida infections involve the formation of biofilms on implanted devices or on tissue surfaces.19,20 C. albicans biofilms consist of matrix-enclosed microcolonies of yeasts and hyphae, and are also capable of holding other microorganisms.21 Detachment of cells from an adherent population can give rise to a septicaemia that may respond to conventional antifungal therapy; biofilm cells are not killed and remain as a reservoir of infection because of their resistance both to host defence mechanisms and several antifungal agents, including fluconazole.18 However, amphotericin B and, especially, the more recent echinocandins, such as caspofungin, are effective in inhibiting the growth of C. albicans biofilms, even though such inhibition occurs at concentrations higher than the MIC concentrations determined according to the NCCLS method.22 EM-01D2 was active against C. albicans biofilms with MIC50 (29.20 mg/L) significantly higher than the MIC50 determined under planktonic growth conditions. More interestingly, the pre-exposure of C. albicans biofilms to subinhibitory concentrations of EM-01D2 led to increased susceptibility to amphotericin B and, consequently, to a significant reduction of MIC50. On the other hand, previous studies demonstrated that pre-exposure of C. albicans to fluconazole or itraconazole generates a subpopulation of cells phenotypically resistant to amphotericin B that can tolerate higher concentrations of amphotericin B than those tolerated by most cells not exposed to azoles.3 An antagonistic effect of high doses of fluconazole with caspofungin was also observed in timekill experiments in C. albicans biofilms.22 These data indicate that the use of azoles, such as fluconazole and itraconazole prophylaxis as a measure to prevent invasive fungal infections, can result in adaptive responses of C. albicans to amphotericin B and other antifungal agents that may have major clinical implications. In particular, in hospitalized patients the treatment of candidiasis associated with intravenous lines and bioprosthetic devices after fluconazole prophylaxis can therefore be problematic.
New antifungal agents are needed because of the importance of fungal infections (in particular in immunocompromised patients), the limitations of some current antifungal agents regarding their toxicity, and the increasing prevalence of drug-resistant isolates. We have demonstrated that EM-01D2, a novel thiazole derivative, had broad spectrum, fungicidal activity and was active against the most clinically relevant yeast. It was also active against C. albicans isolates resistant to fluconazole with MIC and MFC values comparable to those shown by amphotericin B. However, as a consequence of its low toxicity, the IC50/MIC50 and IC50/MFC50 ratios calculated for EM-01D2 were significantly higher than those calculated for amphotericin B. Unlike fluconazole, EM-01D2 was capable of inhibiting the growth of C. albicans biofilms, even if such inhibition occurred at concentrations higher than those determined with planktonic cells. However, the activity of amphotericin B on C. albicans biofilms was significantly increased by pre-exposure to subinhibitory concentrations of EM-01D2. Further studies on the mechanism(s) of action and in vivo efficacy are needed. However, these data indicate the potential of EM-01D2 as a template for the development of new antifungal agents.
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