In vitro effects of promethazine on cell morphology and structure and mitochondrial activity of azole-resistant Candida tropicalis

In vitro effects of promethazine on cell morphology and structure and mitochondrial activity of... Abstract The aim of this study was to evaluate the effect of promethazine on the antifungal minimum inhibitory concentrations against planktonic cells and mature biofilms of Candida tropicalis, as well as investigate its potential mechanisms of cell damage against this yeast species. Three C. tropicalis isolates (two azole-resistant and one azole-susceptible) were evaluated for their planktonic and biofilm susceptibility to promethazine alone and in combination with itraconazole, fluconazole, voriconazole, amphotericin B, and caspofungin. The antifungal activity of promethazine against C. tropicalis was investigated by performing time-kill curve assays and assessing rhodamine 6G efflux, cell size/granularity, membrane integrity, and mitochondrial transmembrane potential, through flow cytometry. Promethazine showed antifungal activity against planktonic cells and biofilms at concentrations of 64 and 128 μg/ml, respectively. The addition of two subinhibitory concentrations of promethazine reduced the antifungal MICs for all tested azole drugs against planktonic growth, reversing the resistance phenotype to all azoles. Promethazine decreased the efflux of rhodamine 6G in an azole-resistant strain. Moreover, promethazine decreased cell size/granularity and caused membrane damage, and mitochondrial membrane depolarization. In conclusion, promethazine presented synergy with azole antifungals against resistant C. tropicalis and exhibited in vitro cytotoxicity against C. tropicalis, altering cell size/granularity, membrane integrity, and mitochondrial function, demonstrating potential mechanisms of cell damage against this yeast species. Promethazine, Candida tropicalis, azole resistance, synergism, biofilm Introduction C. tropicalis is frequently isolated from patients with candidiasis, and it currently is the first or second most frequent non-Candida albicans Candida species involved in cases of candidemia and candiduria.1 Azole resistance in C. tropicalis has been frequently reported,2 especially among clinical isolates,3–5 including those showing cross-resistance to azole antifungals. Azole resistance in C. tropicalis results from the overexpression and/or mutation in the ERG11 gene that encodes the cytochrome P450 lanosterol 14α-demethylase,6 the primary target for azoles. Furthermore, at least two families of multidrug transporters are involved in the development of azole resistance in Candida spp.,7 due to the up-regulation of genes that encode the efflux proteins CDR1 and MDR1.8,9 In addition, alternative mechanisms, such as biofilm formation and mitochondrial defects, have also been reported as strategies to develop azole resistance in Candida spp.10,11 Candida biofilms are one of the main leading causes of infections related to medical devices.12 Biofilm sessile cells are less susceptible to antimicrobial drugs and host defenses, which complicates the treatment of the infection.13 In this context, C. tropicalis biofilms show reduced susceptibility to azole drugs.14 Thus, the search for compounds with antifungal and antibiofilm activities becomes relevant. Promethazine is a first-generation H1 receptor antagonist, with antihistamine and antiemetic effects, which belongs to phenothiazines,15 a group of compounds known to present efflux pump inhibitory properties.16 Some studies have evaluated the antifungal activity of promethazine;17,18 however, further investigations are needed to better understand these inhibitory effects on fungal cells. Thus, the aim of this study was to evaluate the effect of promethazine on the antifungal minimum inhibitory concentrations against planktonic cells and mature biofilms of C. tropicalis, as well as investigate its potential mechanisms of cell damage against this yeast species. Methods Isolates Three C. tropicalis strains, two azole-resistant and one azole-susceptible, from urine, bronchoalveolar lavage, and tracheal aspirate, respectively, were used in this study. These isolates belong to the Culture Collection of the Specialized Medical Mycology Center, Brazil, where they are stored in saline solution at 4°C. Strain identification was based on micromorphological features on cornmeal-Tween 80 agar, carbon and nitrogen assimilation, and carbon fermentation.14 Single colonies were transferred to potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA) for further assays. The phenotypical identification of the isolates was confirmed by matrix-assisted laser desorption-ionization time of flight (MALDI-TOF), as described by Lima-Neto et al.19 The equipment used was MALDI-TOF Autoflex III Mass Spectrometer (Bruker Daltonics Inc., Billerica, MA, USA/Germany), equipped with a Nd:YAG (neodymium-doped yttrium aluminum garnet; Nd:Y3Al5O12) laser of 1064 nm, set to a 66% power. The mass range from 2000 to 20,000 Da was recorded using a linear mode with a delay of 104 ns and an acceleration voltage of +20 kV. The resulting peak lists were exported to the software MALDI Biotyper™ 3.0 (Bruker Daltonics, Bremem, Germany), which provided the final identification of the isolates as C. tropicalis. Antifungal susceptibility testing Minimum inhibitory concentrations (MICs) for amphotericin B, itraconazole, fluconazole, voriconazole, caspofungin, and promethazine were investigated by broth microdilution, according to the document M27-A3.20 In order to determine the susceptibility of the planktonic cells, drugs were tested at concentrations ranging from 0.00390625 to 8 μg/ml for caspofungin, from 0.0625 to 16 μg/ml for amphotericin B, from 0.125 to 1024 for fluconazole, from 0.03125 to 512 μg/ml for itraconazole and voriconazole, and from 1 to 512 μg/ml for promethazine. The maximum or minimum concentration tested was adapted in order to determine the MICs. Serial twofold dilutions of each antifungal were prepared in RPMI 1640 medium with L-glutamine and without sodium bicarbonate (Sigma Chemical Co., St. Louis, MO, USA), buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS; Sigma Chemical Co., USA). Susceptibility testing for planktonic cells was performed in 96-well plates at 35°C for 24 hours. All isolates were tested in duplicate. For amphotericin B, the MIC was defined as the lowest drug concentration that inhibits 100% of fungal growth, while for azoles, caspofungin and promethazine, MICs were defined as the lowest drug concentration capable of inhibiting 50% of fungal growth, when compared to the drug-free growth control.20C. tropicalis ATCC 750 was used as a reference strain, which was kindly given to our research group by Coleção de Microrganismos de Referência em Vigilância Sanitária-CMRVS, FIOCRUZ-INCQS, Rio de Janeiro, RJ, Brazil, where it is maintained under the number INCQS 40281. C. parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were included as quality control for each test. Isolates with MICs ≥1.0 μg/ml, ≥1.0 μg/ml, ≥8.0 μg/ml, ≥1.0 μg/ml were considered resistant to itraconazole, voriconazole, fluconazole and caspofungin, respectively.20,21 Paradoxical growth after 120 h of incubation was confirmed, when a significant increase in cell growth was observed with caspofungin concentrations that were at least two concentrations above the MIC.22 Minimum fungicidal concentration (MFC) for promethazine was determined by seeding the content from all visually clear wells onto PDA plates. The MFC was the lowest drug concentration that killed 99.9% of the fungal inoculums.23 Time-kill curve study Time-kill curve assay was performed in standard RPMI medium, according to the method described by Cantón et al.,23 with modifications. Previously, the isolates were subcultured on PDA and grown at 35°C, for 24 h. The inoculum was adjusted to the density of 0.5 on McFarland's turbidity standard. The adjusted inoculum was diluted 1:20 in RPMI containing promethazine at 16, 32, 64, or 128μg/ml, with a final volume of 5 ml. This procedure yielded an initial inoculum of 4–6 × 104 cfu/ml. Then, the fungal suspensions were incubated at 35°C without agitation. At seven predetermined time points (0, 2, 4, 6, 12, 24, and 48 h), a 0.1-ml aliquot was removed from the drug-free control tube and each tube containing the test solution and serially diluted in sterile saline, reaching up to 6 dilutions, depending on the amount of fungal growth and drug concentration. Aliquots of 0.1 ml were spread onto PDA plates and incubated at 35°C for 24 to 48 h to determine the number of colony-forming unit per milliliter. Time-kill assays were conducted in duplicate. Biofilm susceptibility testing For the evaluation of antifungal and promethazine susceptibility of sessile cells, biofilms were prepared as described by Brilhante et al.14 Yeast suspensions were adjusted to reach 1 × 106 cells/mL in RPMI medium and then 200 μl were transferred to flat bottomed 96-well polystyrene plates. After 24 h of biofilm formation at 37°C, the medium was discarded and each well was washed three times with sterile phosphate-buffered saline (PBS). Afterward, 200 μl of RPMI 1640 medium containing serial twofold dilutions of each tested drug were added to the wells containing mature C. tropicalis biofilms and the plates were incubated at 37°C for further 48 hours. The drugs were tested at the following concentration ranges: 0.5–512 μg/ml for fluconazole; 0.125–128 μg/ml for itraconazole, voriconazole, amphotericin B; 0.0625–64 μg/ml for caspofungin and 4–2048 μg/ml for promethazine. For each tested strain, drug-free biofilm growth (growth control) and biofilm-free wells (sterility control) were included. Finally, biofilm metabolic activity was quantified using the XTT-reduction assay. Aliquots of 100 μl of XTT menadione solution [0.1 mg/ml XTT, 1 mM menadione (Sigma Chemical Co.)] were added to each well, and the plates were incubated in the dark for 2 h at 37°C, followed by spectrophotometric readings at 492 nm with a microtiter plate reader. Antifungal sessile minimum inhibitory concentrations (SMIC) against biofilm cells were determined as the concentration able of causing 50% (SMIC50) and 90% (SMIC90) inhibition of biofilm metabolic activity, when compared to the metabolic activity of the drug-free biofilm growth control. For caspofungin, the occurrence of paradoxical growth was considered when a metabolic activity of 20% or more of that obtained for the drug-free growth control was observed at drug concentrations above the SMIC.14 All assays were carried out in duplicate. Evaluation of drug interaction For the evaluation of the interaction between promethazine and the antifungal drugs, two sub-inhibitory concentrations of promethazine (16 and 8 μg/ml) were used in the analyses of each isolate. The antifungal susceptibility analysis of planktonic and sessile cells was performed, as previously described, with amphotericin B, caspofungin, fluconazole, itraconazole, and voriconazole. Planktonic and sessile cells were incubated in the presence of the combination of promethazine and antifungals and incubated as above described. C. tropicalis ATCC 750 was also included as reference control strain. Afterward, the fractional inhibitory concentration index (FICI) was calculated for each promethazine combination, in order to evaluate drug interaction, which was considered to be synergistic when FICI ≤ 0.5, indifferent when 0.5 < FICI ≤ 4, and antagonistic when FICI > 4.17,24 Confocal laser scanning microscopy (CLSM) Biofilms of C. tropicalis were analyzed by CLSM, according to Brilhante et al.14 Biofilms were formed directly on ThermanoxTM coverslips, as described above, and different promethazine concentrations (32, 64, 128, 256, 512, 1024, and 2048 μg/ml) were added to the samples. Biofilms were incubated for additional 48 h at 37°C. Control (unexposed) samples were incubated in RPMI 1640 only. After incubation, the ThermanoxTM coverslips containing the adhered biofilm was transferred to a glass bottom Petri dish, where Live/Dead (Invitrogen TM151) fluorescent dye was added to cover the surface of the ThermanoxTM coverslips. Afterward, the coverslips were evaluated under a Confocal Microscope (Nikon C2), at 488 nm for the detection of the SYTO 9 fluorescent dye (live cells) and at 561 nm for the detection of the propidium iodide (dead cells).14 For image analyses, five equidistant points of the coverlips were submitted to Z-stacked capture of the images, for three-dimensional image. Scanning electron microscopy (SEM) Biofilms of C. tropicalis were analyzed by SEM, as described in the previous section (CLSM). After incubation, biofilm supernatants were removed, and slides were washed with cacodylate buffer (0.15 M) twice. Biofilms were then covered with glutaraldehyde (2.5% in cacodylate buffer) and incubated at 4°C overnight. After incubation, biofilms were washed twice with cacodylate buffer, and slides were dehydrated in ascending ethanol concentrations (50, 70, 80, 95, and 100%) twice, for 10 minutes each concentration. Slides were dried at room temperature and covered with hexamethyldisilazane for 30 minutes. Slides were dried overnight, coated with 10 nm of gold (Emitech Q150T), and observed with a FEI Inspect S50 scanning electron microscope, in the high vacuum mode at 15 kV, and images were processed with Photoshop (Adobe Systems, San Jose, CA, USA) software.14 Analysis of efflux pump activity by flow cytometry The activity of efflux pumps was evaluated through flow cytometry by measuring the efflux of rhodamine 6G (R6G), as described by Jiang et al.6 and Ivnitski-Steele et al.,25 with modifications. Yeast cells (106) in YEPD broth (1% yeast extract, 2% peptone, 2% dextrose) were suspended in 2 ml of PBS and incubated for 4 h at 30°C with constant shaking to induce starvation. Starved cells were pre-incubated with promethazine at 8, 16, and 32 μg/ml, for 1 h. After incubation, 15 μM R6G (7.5 mM stock in DMSO) were added and incubated at 30°C, for 20 min, in the dark. Then cells were washed with cold PBS, followed by the addition of glucose (20 mM in PBS) and incubation at 30°C for 30 min. Afterward, cells were washed twice with cold sterile PBS. The fluorescence of each condition was immediately quantified using a FACSCalibur flow cytometer (BD Biosciences), with measurement of 10,000 cells per sample. Each assay was performed in triplicate and an azole-resistant C. parapsilosis, which is known to have an enhanced efflux activity,26 was used as a positive control. Cellular debris was omitted from the analysis. Determination of cell size/granulariry and membrane integrity Cell size and granularity and membrane integrity of the fungal cells were evaluated by flow cytometry. Fungal strains were exposed to promethazine (64, 128, and 256 μg/ml), amphotericin B (control drug, 2 μg/ml), and fluconazole (control drug, 64 μg/ml) in RPMI medium at a final concentration of 106 cells/ml at 35°C, for 2 and 4 h. Then, the cells were washed with PBS and incubated with 2 μg/ml propidium iodide (PI), in the dark, for 30 min. The cellular fluorescence was then determined using flow cytometry, in a FACSCalibur flow cytometer. Each assay was performed in triplicate. A total of 10,000 cells were evaluated per assay, with the cellular debris omitted from the analysis.27 Measurement of mitochondrial transmembrane potential (Δψm) The mitochondrial transmembrane potential was determined by the retention of rhodamine 123 (Rho123) dye by fungal cells, after exposure to promethazine (64, 128, and 256 μg/ml), amphotericin B (control drug, 2 μg/ml), and fluconazole (control drug, 64 μg/ml) in RPMI medium at a final concentration of 106 cells/ml at 35°C, for 2 and 4 h. After exposure, the cells were washed with PBS incubated with 5 μg/ml Rho123, in the dark, at 37°C for 30 min, and then washed twice with PBS. Their fluorescence was measured using flow cytometry, in a FACSCalibur flow cytometer. Each assay was performed in triplicate. A total of 10,000 cells were evaluated per experiment, and cellular debris was omitted from the analysis.27 Statistical analysis All tests were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. P-values < .05 were considered statistically significant. The statistical analyses were performed with GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). Results Results regarding antifungal susceptibility tests are shown in Table 1. Promethazine MIC and MFC were 64 μg/ml and 256 μg/ml, respectively. Fluconazole MICs against azole-resistant isolates were 256 and 512 μg/ml, voriconazole MICs were 32 and 64 μg/ml, and those for itraconazole were 128 μg/ml. Antifungal MICs against the azole-susceptible strain were 0.5, 0.0625, and 0.0625 μg/ml for fluconazole, itraconazole, and voriconazole, respectively. Amphotericin B and caspofungin MIC ranges were 0.25–1 μg/ml and 0.0078–0.0156 μg/ml, respectively. Table 1. Minimum inhibitory concentrations of promethazine and antifungal drugs isolated and in combination with promethazine against Candida tropicalis isolates.   MIC (μg/ml)  FICI    Azole-resistant      Azole-resistant        strains      strains            Azole-susceptible  ATCC 750      Azole-susceptible  ATCC 750  Drugs  Strain 1  Strain 2  strain    Strain 1  Strain 2  strain    PRM  64  64  64            256 (MFC)  256 (MFC)  256 (MFC)  -  -  -  AMB  0.5  1  0.25  0.25  -  -  -  -  AMB+PRM 8  0.125  0.125  0.0625  0.0625  0.375S  0.250S  0.325S  0.325S  AMB+PRM 16  0.125  0.0625  0.0625  0.0625  0.5S  0.312S  0.5S  0.5S  CAS*  0.0156  0.0078  0.0078  0.03125  -  -  -  -  CAS+PRM 8  0.0078  0.0039  0.0078  0.03125  0.625I  0.625I  1.125I  1.125I  CAS+PRM 16  0.0078  0.0039  0.0039  0.0156  0.75I  0.75I  0.75I  0.75I  FLC  512  256  0.5  0.5  -  -  -  -  FLC+PRM 8  4  4  4  4  0.133S  0.141S  8.125A  8.125A  FLC+PRM 16  4  4  2  4  0.258S  0.266S  4.25A  8.25A  ITC  128  128  0.0625  0.0625  -  -  -  -  ITC+PRM 8  0.0625  0.125  0.125  0.03125  0.125S  0.126S  2.125I  0.625I  ITC+PRM 16  0.03125  0.0625  0.03125  0.0156  0.25S  0.25S  0.75I  0.5S  VRC  32  64  0.0625  0.03125  -  -  -  -  VRC+PRM 8  0.5  1  0.25  0.25  0.141S  0.141S  4.125A  2.125I  VRC+PRM 16  0.25  0.5  0.25  0.125  0.258S  0.258S  4.25A  4.25A    MIC (μg/ml)  FICI    Azole-resistant      Azole-resistant        strains      strains            Azole-susceptible  ATCC 750      Azole-susceptible  ATCC 750  Drugs  Strain 1  Strain 2  strain    Strain 1  Strain 2  strain    PRM  64  64  64            256 (MFC)  256 (MFC)  256 (MFC)  -  -  -  AMB  0.5  1  0.25  0.25  -  -  -  -  AMB+PRM 8  0.125  0.125  0.0625  0.0625  0.375S  0.250S  0.325S  0.325S  AMB+PRM 16  0.125  0.0625  0.0625  0.0625  0.5S  0.312S  0.5S  0.5S  CAS*  0.0156  0.0078  0.0078  0.03125  -  -  -  -  CAS+PRM 8  0.0078  0.0039  0.0078  0.03125  0.625I  0.625I  1.125I  1.125I  CAS+PRM 16  0.0078  0.0039  0.0039  0.0156  0.75I  0.75I  0.75I  0.75I  FLC  512  256  0.5  0.5  -  -  -  -  FLC+PRM 8  4  4  4  4  0.133S  0.141S  8.125A  8.125A  FLC+PRM 16  4  4  2  4  0.258S  0.266S  4.25A  8.25A  ITC  128  128  0.0625  0.0625  -  -  -  -  ITC+PRM 8  0.0625  0.125  0.125  0.03125  0.125S  0.126S  2.125I  0.625I  ITC+PRM 16  0.03125  0.0625  0.03125  0.0156  0.25S  0.25S  0.75I  0.5S  VRC  32  64  0.0625  0.03125  -  -  -  -  VRC+PRM 8  0.5  1  0.25  0.25  0.141S  0.141S  4.125A  2.125I  VRC+PRM 16  0.25  0.5  0.25  0.125  0.258S  0.258S  4.25A  4.25A  Strain 1: azole-resistant strain with enhanced efflux activity. Strain 2: azole-resistant strain with decreased efflux activity. AMB, amphotericin B; CAS, caspofungin; FICI, fractional inhibitory concentration index; FLC, fluconazole; ITC, itraconazole; MFC, minimum fungicidal concentration; MIC, minimum inhibitory concentration; PRM, promethazine; PRM 8, promethazine at 8 μg/ml; PRM 16, promethazine at 16 μg/ml; VRC, voriconazole. Ssynergistic interaction (FICI ≤ 0.5); I indifferent interaction (0.5 > FICI ≤ 4); A antagonistic interaction (FICI > 4). *Paradoxical growth effect of caspofungin alone was observed at 8 μg/ml only against azole susceptible isolate, and at 4 to 16 μg/ml when combined with promethazine (8 and 16 μg/ml) against azole susceptible and resistant C. tropicalis isolates. View Large Table 1. Minimum inhibitory concentrations of promethazine and antifungal drugs isolated and in combination with promethazine against Candida tropicalis isolates.   MIC (μg/ml)  FICI    Azole-resistant      Azole-resistant        strains      strains            Azole-susceptible  ATCC 750      Azole-susceptible  ATCC 750  Drugs  Strain 1  Strain 2  strain    Strain 1  Strain 2  strain    PRM  64  64  64            256 (MFC)  256 (MFC)  256 (MFC)  -  -  -  AMB  0.5  1  0.25  0.25  -  -  -  -  AMB+PRM 8  0.125  0.125  0.0625  0.0625  0.375S  0.250S  0.325S  0.325S  AMB+PRM 16  0.125  0.0625  0.0625  0.0625  0.5S  0.312S  0.5S  0.5S  CAS*  0.0156  0.0078  0.0078  0.03125  -  -  -  -  CAS+PRM 8  0.0078  0.0039  0.0078  0.03125  0.625I  0.625I  1.125I  1.125I  CAS+PRM 16  0.0078  0.0039  0.0039  0.0156  0.75I  0.75I  0.75I  0.75I  FLC  512  256  0.5  0.5  -  -  -  -  FLC+PRM 8  4  4  4  4  0.133S  0.141S  8.125A  8.125A  FLC+PRM 16  4  4  2  4  0.258S  0.266S  4.25A  8.25A  ITC  128  128  0.0625  0.0625  -  -  -  -  ITC+PRM 8  0.0625  0.125  0.125  0.03125  0.125S  0.126S  2.125I  0.625I  ITC+PRM 16  0.03125  0.0625  0.03125  0.0156  0.25S  0.25S  0.75I  0.5S  VRC  32  64  0.0625  0.03125  -  -  -  -  VRC+PRM 8  0.5  1  0.25  0.25  0.141S  0.141S  4.125A  2.125I  VRC+PRM 16  0.25  0.5  0.25  0.125  0.258S  0.258S  4.25A  4.25A    MIC (μg/ml)  FICI    Azole-resistant      Azole-resistant        strains      strains            Azole-susceptible  ATCC 750      Azole-susceptible  ATCC 750  Drugs  Strain 1  Strain 2  strain    Strain 1  Strain 2  strain    PRM  64  64  64            256 (MFC)  256 (MFC)  256 (MFC)  -  -  -  AMB  0.5  1  0.25  0.25  -  -  -  -  AMB+PRM 8  0.125  0.125  0.0625  0.0625  0.375S  0.250S  0.325S  0.325S  AMB+PRM 16  0.125  0.0625  0.0625  0.0625  0.5S  0.312S  0.5S  0.5S  CAS*  0.0156  0.0078  0.0078  0.03125  -  -  -  -  CAS+PRM 8  0.0078  0.0039  0.0078  0.03125  0.625I  0.625I  1.125I  1.125I  CAS+PRM 16  0.0078  0.0039  0.0039  0.0156  0.75I  0.75I  0.75I  0.75I  FLC  512  256  0.5  0.5  -  -  -  -  FLC+PRM 8  4  4  4  4  0.133S  0.141S  8.125A  8.125A  FLC+PRM 16  4  4  2  4  0.258S  0.266S  4.25A  8.25A  ITC  128  128  0.0625  0.0625  -  -  -  -  ITC+PRM 8  0.0625  0.125  0.125  0.03125  0.125S  0.126S  2.125I  0.625I  ITC+PRM 16  0.03125  0.0625  0.03125  0.0156  0.25S  0.25S  0.75I  0.5S  VRC  32  64  0.0625  0.03125  -  -  -  -  VRC+PRM 8  0.5  1  0.25  0.25  0.141S  0.141S  4.125A  2.125I  VRC+PRM 16  0.25  0.5  0.25  0.125  0.258S  0.258S  4.25A  4.25A  Strain 1: azole-resistant strain with enhanced efflux activity. Strain 2: azole-resistant strain with decreased efflux activity. AMB, amphotericin B; CAS, caspofungin; FICI, fractional inhibitory concentration index; FLC, fluconazole; ITC, itraconazole; MFC, minimum fungicidal concentration; MIC, minimum inhibitory concentration; PRM, promethazine; PRM 8, promethazine at 8 μg/ml; PRM 16, promethazine at 16 μg/ml; VRC, voriconazole. Ssynergistic interaction (FICI ≤ 0.5); I indifferent interaction (0.5 > FICI ≤ 4); A antagonistic interaction (FICI > 4). *Paradoxical growth effect of caspofungin alone was observed at 8 μg/ml only against azole susceptible isolate, and at 4 to 16 μg/ml when combined with promethazine (8 and 16 μg/ml) against azole susceptible and resistant C. tropicalis isolates. View Large The tested subinhibitory concentrations of promethazine (8 and 16 μg/ml) reduced the antifungal MICs and reversed the azole resistance phenotype of the two azole-resistant isolates. The MICs against the azole-resistant isolates reduced 64- to 128-fold for fluconazole and voriconazole and 1024- to 4096-fold for itraconazole (Table 1). However, no synergistic interaction was observed for the combination of promethazine and azoles against the azole-susceptible strain and the reference strain (C. tropicalis ATCC 750), and antagonism was observed when promethazine was combined with fluconazole and voriconazole. Promethazine reduced the MICs for amphotericin B and caspofungin two- to 16-fold against the three C. tropicalis isolates and the reference strain. Paradoxical growth effect of caspofungin alone was only observed against the azole susceptible isolate, at 8 μg/ml. As for the combination of caspofungin and subinhibitory concentrations of promethazine (8 and 16 μg/ml), paradoxical growth was observed at 4 to 16 μg/ml of caspofungin against azole susceptible and resistant isolates (Table 1). The killing activity of promethazine against C. tropicalis is shown in Figure 1. Reduction in the number of cfu per milliliter was observed after 2 h of incubation of the yeast cells at different concentrations of the drug. At concentrations of 64, 128, and 256 μg/ml, the time required to kill 50% of the initial inoculum was 2 h. At the concentration four times the MIC (256 μg/ml), 99% reduction of live fungal cells was observed after 4 h of incubation, and no viable cells were observed after 12 h of incubation. Figure 1. View largeDownload slide Representative plots of mean ± standard deviation of colony-forming unit counts for the establishment of a time-kill curve for Candida tropicalis after exposure to promethazine at 32 μg/ml (square), 64 μg/ml (diamond), 128 μg/ml (circle), and 256 μg/ml (×). Drug-free growth control was also added for comparative purposes (triangle). Figure 1. View largeDownload slide Representative plots of mean ± standard deviation of colony-forming unit counts for the establishment of a time-kill curve for Candida tropicalis after exposure to promethazine at 32 μg/ml (square), 64 μg/ml (diamond), 128 μg/ml (circle), and 256 μg/ml (×). Drug-free growth control was also added for comparative purposes (triangle). Regarding mature biofilm susceptibility, promethazine showed SMIC50 of 128 μg/ml and SMIC90 of 256 μg/ml, 2 times and 4 times the MIC for planktonic cells, respectively (Table 2). Antifungal SMIC50 and SMIC90 against C. tropicalis mature biofilm were 8 and 32 μg/ml for amphotericin B, <0.0625 μg/ml for caspofungin, >512 μg/ml for fluconazole and >128 μg/ml for itraconazole and voriconazole. Promethazine did not significantly modify the biofilm susceptibility to azoles, amphotericin B and caspofungin (Table 2). The minimum caspofungin concentration that induced paradoxical biofilm growth decreased four- to eightfold in the presence of promethazine (Table 2). Table 2. Sessile minimum inhibitory concentrations of promethazine and antifungal drugs isolated and in combination with promethazine against Candida tropicalis mature biofilms Drugs  SMIC50  SMIC90    (μg/ml)  (μg/ml)  PRM  128  256  AMB  8  32  AMB+PRM 8  16  64  AMB+PRM 16  16  64  CAS*  <0.0625  <0.0625  CAS+PRM 8*  <0.0625  <0.0625  CAS+PRM16*  <0.0625  <0.0625  FLC  >512  >512  FLC+PRM 8  >512  >512  FLC+PRM 16  >512  >512  ITC  >128  >128  ITC+PRM 8  >128  >128  ITC+PRM 16  >128  >128  VRC  >128  >128  VRC+PRM 8  >128  >128  VRC+PRM 16  >128  >128  Drugs  SMIC50  SMIC90    (μg/ml)  (μg/ml)  PRM  128  256  AMB  8  32  AMB+PRM 8  16  64  AMB+PRM 16  16  64  CAS*  <0.0625  <0.0625  CAS+PRM 8*  <0.0625  <0.0625  CAS+PRM16*  <0.0625  <0.0625  FLC  >512  >512  FLC+PRM 8  >512  >512  FLC+PRM 16  >512  >512  ITC  >128  >128  ITC+PRM 8  >128  >128  ITC+PRM 16  >128  >128  VRC  >128  >128  VRC+PRM 8  >128  >128  VRC+PRM 16  >128  >128  SMIC50: sessile minimum inhibitory concentration that causes 50% inhibition of biofilm metabolic activity, measured through XTT reduction assay; SMIC90: sessile minimum inhibitory concentration that causes 90% inhibition of biofilm metabolic activity, measured through XTT reduction assay. AMB, amphotericin B; CAS, caspofungin; FLC, fluconazole; ITC, itraconazole; PRM, promethazine; PRM 8, promethazine at 8 μg/ml; PRM 16, promethazine at 16 μg/ml; VRC: voriconazole. *Paradoxical growth effect of caspofungin alone was observed at 4 μg/ml, and at 1 and 0.5 μg/ml, when combined with promethazine at 8 and 16 μg/ml, respectively. View Large Table 2. Sessile minimum inhibitory concentrations of promethazine and antifungal drugs isolated and in combination with promethazine against Candida tropicalis mature biofilms Drugs  SMIC50  SMIC90    (μg/ml)  (μg/ml)  PRM  128  256  AMB  8  32  AMB+PRM 8  16  64  AMB+PRM 16  16  64  CAS*  <0.0625  <0.0625  CAS+PRM 8*  <0.0625  <0.0625  CAS+PRM16*  <0.0625  <0.0625  FLC  >512  >512  FLC+PRM 8  >512  >512  FLC+PRM 16  >512  >512  ITC  >128  >128  ITC+PRM 8  >128  >128  ITC+PRM 16  >128  >128  VRC  >128  >128  VRC+PRM 8  >128  >128  VRC+PRM 16  >128  >128  Drugs  SMIC50  SMIC90    (μg/ml)  (μg/ml)  PRM  128  256  AMB  8  32  AMB+PRM 8  16  64  AMB+PRM 16  16  64  CAS*  <0.0625  <0.0625  CAS+PRM 8*  <0.0625  <0.0625  CAS+PRM16*  <0.0625  <0.0625  FLC  >512  >512  FLC+PRM 8  >512  >512  FLC+PRM 16  >512  >512  ITC  >128  >128  ITC+PRM 8  >128  >128  ITC+PRM 16  >128  >128  VRC  >128  >128  VRC+PRM 8  >128  >128  VRC+PRM 16  >128  >128  SMIC50: sessile minimum inhibitory concentration that causes 50% inhibition of biofilm metabolic activity, measured through XTT reduction assay; SMIC90: sessile minimum inhibitory concentration that causes 90% inhibition of biofilm metabolic activity, measured through XTT reduction assay. AMB, amphotericin B; CAS, caspofungin; FLC, fluconazole; ITC, itraconazole; PRM, promethazine; PRM 8, promethazine at 8 μg/ml; PRM 16, promethazine at 16 μg/ml; VRC: voriconazole. *Paradoxical growth effect of caspofungin alone was observed at 4 μg/ml, and at 1 and 0.5 μg/ml, when combined with promethazine at 8 and 16 μg/ml, respectively. View Large Alterations in biofilm structure and morphological changes in biofilm-associated cells after exposure to different promethazine concentrations were observed using CLSM (Fig. 2) and SEM (Fig. 3). Fragmentation and reduction of viable cells and biomass were observed when biofilms were exposed to concentrations starting at 32 μg/ml of promethazine (MIC/2 against planktonic cells). Decrease in biofilm thickness was observed in all tested concentrations (Fig. 2). Biomass increased at higher concentrations (1024 and 2048 μg/ml, Fig. 2 and 3); however, no live cells were observed (Fig. 2). In addition, morphological changes in yeast cells were observed at 256 and 512 μg/ml, showing collapsed cells with wrinkled surface (Fig. 3). Figure 2. View largeDownload slide Confocal laser scanning microscopy images of Candida tropicalis mature biofilms after 48 h of incubation at different concentrations of promethazine (PRM). Notice the decrease in biofilm thickness and in the number of live cells at all tested concentrations, when compared to growth control. An increase in biomass and biofilm thickness is observed at the highest concentrations, however no live cells are detected. Magnification: 400 ×. Lasers: 488 nm (images a–d, SYTO9, viable cells), 561 nm (images e–h, propidium iodide, dead or damaged cells). Scale: 200 μm. Figure 2. View largeDownload slide Confocal laser scanning microscopy images of Candida tropicalis mature biofilms after 48 h of incubation at different concentrations of promethazine (PRM). Notice the decrease in biofilm thickness and in the number of live cells at all tested concentrations, when compared to growth control. An increase in biomass and biofilm thickness is observed at the highest concentrations, however no live cells are detected. Magnification: 400 ×. Lasers: 488 nm (images a–d, SYTO9, viable cells), 561 nm (images e–h, propidium iodide, dead or damaged cells). Scale: 200 μm. Figure 3. View largeDownload slide Scanning electron microscopy of Candida tropicalis mature biofilm after 48 h of incubation at different concentrations of promethazine (PRM). (a) drug-free growth control; (b) PRM 64 μg/ml, notice a reduced number of cells and loss of biofilm structure; (c) PRM 256 μg/ml, notice the increased number of morphologically altered cells (cells with empty appearance, arrows) and disrupted biofilm; (d) PRM 2048 μg/ml; notice an increase in biomass and presence of matrix remnants, with microscopically altered cells (wrinkled cells, arrows). Magnification: 5000 ×. Scale: 20 μm. Figure 3. View largeDownload slide Scanning electron microscopy of Candida tropicalis mature biofilm after 48 h of incubation at different concentrations of promethazine (PRM). (a) drug-free growth control; (b) PRM 64 μg/ml, notice a reduced number of cells and loss of biofilm structure; (c) PRM 256 μg/ml, notice the increased number of morphologically altered cells (cells with empty appearance, arrows) and disrupted biofilm; (d) PRM 2048 μg/ml; notice an increase in biomass and presence of matrix remnants, with microscopically altered cells (wrinkled cells, arrows). Magnification: 5000 ×. Scale: 20 μm. The uptake and efflux of R6G were quantified using flow cytometry to evaluate the activity of yeast efflux pumps and the effect of promethazine on this mechanism of survival and drug resistance. Initially, the efflux activity of the strains was assessed and one azole-resistant isolate showed significant decrease in intracellular fluorescence in the presence of glucose, when compared to the cells that were submitted to starvation (P < .05), demonstrating the efflux activity of this strain. Control strain (C. parapsilosis with enhanced efflux activity) also showed significant decrease in intracellular fluorescence in the presence of glucose in comparison with cells in starvation (P < .05). Afterward, the effect of promethazine was evaluated. Preincubation with promethazine (8, 16, and 32 μg/ml) induced an increase in the mean intracellular fluorescence intensity of the azole-resistant isolate that showed efflux activity, when compared to the unexposed control in the presence of glucose (P < .05). However, for the azole-resistant strain that did not show efflux activity and for the azole-susceptible strain, preincubation with promethazine caused a little but significant decrease in the mean fluorescence intensity of the cells, when compared to the cells in starvation and the unexposed control in the presence of glucose (P < .05). Promethazine also induced a slight but significant increase in the mean intracellular fluorescence intensity of the control strain (P < .05). A decrease in cell size/granularity was observed after 2 and 4 h incubation of C. tropicalis strains with promethazine at MIC, 2 × MIC, and 4 × MIC, similar to what was observed with amphotericin B (Fig. 4). As shown in Figure 5, a significant increase in the number of cells with plasma membrane damage was observed when yeast cells were exposed to different concentrations of promethazine, after 2 and 4 h of incubation, as demonstrated by the propidium iodide assay. Moreover, significant changes in the mitochondrial transmembrane potential were observed after 2 and 4 h of exposure to promethazine (Table 3). Rh123 fluorescence was lower in yeast cells exposed to promethazine, when compared to unexposed cells (P < .05). Decrease in Rh123 fluorescence was also observed when cells were exposed to amphotericin B. Figure 4. View largeDownload slide Representative flow cytometry analysis of changes in size (forward scatter, FSC) and granularity (side scatter, SSC) obtained for the azole-resistant (n = 2) and azole-susceptible (n = 1) strains of Candida tropicalis, after 4 h of exposure to promethazine (PRM), fluconazole (FLC) and amphotericin B (AMB). (a) untreated control; (b) FLC 64 μg/ml; (c) AMB 2 μg/ml; (d) PRM 64 μg/ml; (e) PRM 128 μg/ml; (f) PRM 256 μg/ml. Results are shown in mean fluorescence intensity of cells. Dashed lines represent cellular fluorescence of the untreated control (a). Reduction in cellular fluorescence indicates reduction in cells size (FSC) and/or granularity (SSC). Notice a reduction in cell size and granularity when cells are exposed to PRM and AMB, which is common for dying cells. Figure 4. View largeDownload slide Representative flow cytometry analysis of changes in size (forward scatter, FSC) and granularity (side scatter, SSC) obtained for the azole-resistant (n = 2) and azole-susceptible (n = 1) strains of Candida tropicalis, after 4 h of exposure to promethazine (PRM), fluconazole (FLC) and amphotericin B (AMB). (a) untreated control; (b) FLC 64 μg/ml; (c) AMB 2 μg/ml; (d) PRM 64 μg/ml; (e) PRM 128 μg/ml; (f) PRM 256 μg/ml. Results are shown in mean fluorescence intensity of cells. Dashed lines represent cellular fluorescence of the untreated control (a). Reduction in cellular fluorescence indicates reduction in cells size (FSC) and/or granularity (SSC). Notice a reduction in cell size and granularity when cells are exposed to PRM and AMB, which is common for dying cells. Figure 5. View largeDownload slide Representative analysis of mean fluorescence intensity of propidium iodide obtained for the azole-resistant (n = 2) and azole-susceptible (n = 1) strains of Candida tropicalis, following exposure to promethazine after 2 (a) and 4h (b) of incubation. Control: unexposed cells. AMB, amphotericin B. FLC, fluconazole. PRM, promethazine. Increase in intracellular fluorescence indicates plasma membrane damage. *indicates significant differences when compared to the unexposed control. Each bar represents the mean ± SD of triplicate readings of one representative strain. Figure 5. View largeDownload slide Representative analysis of mean fluorescence intensity of propidium iodide obtained for the azole-resistant (n = 2) and azole-susceptible (n = 1) strains of Candida tropicalis, following exposure to promethazine after 2 (a) and 4h (b) of incubation. Control: unexposed cells. AMB, amphotericin B. FLC, fluconazole. PRM, promethazine. Increase in intracellular fluorescence indicates plasma membrane damage. *indicates significant differences when compared to the unexposed control. Each bar represents the mean ± SD of triplicate readings of one representative strain. Table 3. Mean fluorescence intensity of rhodamine 123 (Rh123) in azole-resistant (n = 2) and azole-susceptible (n = 1) Candida tropicalis strains following exposure to promethazine.         PRM  Time of incubation  Control  FLC  AMB  64 μg/ml  128 μg/ml  256 μg/ml  2 h  72.85 ± 20.11  705.2 ± 202.1*  10.38 ± 2.01*  18.33 ± 5.28*  10.99 ± 2.56*  8.9 ± 0.59*  4 h  72.93 ± 14.71  1543 ± 11.86*  12.5 ± 1.18*  33 ± 6.79*  11.32 ± 0.86*  20.57 ± 9.67*          PRM  Time of incubation  Control  FLC  AMB  64 μg/ml  128 μg/ml  256 μg/ml  2 h  72.85 ± 20.11  705.2 ± 202.1*  10.38 ± 2.01*  18.33 ± 5.28*  10.99 ± 2.56*  8.9 ± 0.59*  4 h  72.93 ± 14.71  1543 ± 11.86*  12.5 ± 1.18*  33 ± 6.79*  11.32 ± 0.86*  20.57 ± 9.67*  Data represent mean fluorescence intensity. Control: untreated cells. AMB, amphotericin B 2 μg/ml. FLC, fluconazole 64 μg/ml. PRM, promethazine. Decrease in Rh123 fluorescence indicates depolarization and de-energization of mitochondria and impaired mitochondrial function. *significant difference when compared to untreated control. Data are expressed as mean ± SD of triplicate readings per tested strain. View Large Table 3. Mean fluorescence intensity of rhodamine 123 (Rh123) in azole-resistant (n = 2) and azole-susceptible (n = 1) Candida tropicalis strains following exposure to promethazine.         PRM  Time of incubation  Control  FLC  AMB  64 μg/ml  128 μg/ml  256 μg/ml  2 h  72.85 ± 20.11  705.2 ± 202.1*  10.38 ± 2.01*  18.33 ± 5.28*  10.99 ± 2.56*  8.9 ± 0.59*  4 h  72.93 ± 14.71  1543 ± 11.86*  12.5 ± 1.18*  33 ± 6.79*  11.32 ± 0.86*  20.57 ± 9.67*          PRM  Time of incubation  Control  FLC  AMB  64 μg/ml  128 μg/ml  256 μg/ml  2 h  72.85 ± 20.11  705.2 ± 202.1*  10.38 ± 2.01*  18.33 ± 5.28*  10.99 ± 2.56*  8.9 ± 0.59*  4 h  72.93 ± 14.71  1543 ± 11.86*  12.5 ± 1.18*  33 ± 6.79*  11.32 ± 0.86*  20.57 ± 9.67*  Data represent mean fluorescence intensity. Control: untreated cells. AMB, amphotericin B 2 μg/ml. FLC, fluconazole 64 μg/ml. PRM, promethazine. Decrease in Rh123 fluorescence indicates depolarization and de-energization of mitochondria and impaired mitochondrial function. *significant difference when compared to untreated control. Data are expressed as mean ± SD of triplicate readings per tested strain. View Large Discussion Azole resistance in C. tropicalis has become more common in the past years;4,5,28 therefore, the quest for alternatives to overcome this obstacle has become relevant. In the present study, promethazine, a compound derived from phenothiazine, was evaluated against planktonic and sessile cells of azole-resistant C. tropicalis. The azole-resistance phenotype was altered when C. tropicalis isolates were treated with subinhibitory concentrations of promethazine, with azole MIC reductions reaching up to 2048-fold. Similar results were observed by Castelo-Branco et al.17 and Brilhante et al.29 who tested promethazine in combination with azole antifungals against azole-resistant Candida species, showing synergistic interactions. The authors suggested that this synergism was due to efflux pump inhibition by promethazine, an important mechanism of resistance in Candida species. Iatta et al.30 suggested that the synergistic effect of promethazine and azoles against Malassezia species might be related to an increased efflux pump activity. This also seems to be the mechanism of drug interaction against C. tropicalis strains, since synergistic interactions between azoles and promethazine were only observed with azole-resistant isolates. Also, MIC reductions for amphotericin B and caspofungin were significant lower than those observed for azoles. Thus, possible mechanisms of cell damage caused by promethazine were assessed in order to better understand the interaction between promethazine and azole antifungals. The time-kill assay demonstrated that cell death is observed as soon as 2 h after incubation with promethazine at MIC, 2 × MIC and 4 × MIC. Cell death rate increases as drug concentration increases, reaching 100% death at 4 times the MIC (256 μg/ml), after 12 h of incubation, in agreement with the MFC results. This result indicates a possible fungicidal mechanism of promethazine against C. tropicalis.23 The mechanism of cell death of promethazine against C. tropicalis may be partially caused by damaged mitochondrial function, altered cell respiration and changes in cell structure, mainly membrane damage. Promethazine inhibited mature biofilms at concentrations twice the MIC for planktonic cells, showing an effective antibiofilm activity. Promethazine also promoted significant alterations in biofilm structure, decreasing the amount of hyphae and reducing its thickness. This structural change was more noticeable at lower concentrations of promethazine, while an increase in biomass was observed at the highest tested concentrations, although, live cells were not observed. The antibiofilm effects of promethazine alone may be of particular therapeutic interest, since biofilms are commonly associated with Candida infections and are inherently difficult to treat.12 On the other hand, promethazine did not reduce the antifungal inhibitory concentrations against C. tropicalis mature biofilms, contrasting the results obtained against planktonic cells. Moreover, paradoxical growth effect of caspofungin on planktonic and sessile cells was detected at lower caspofungin concentration, when this antifungal was combined with promethazine. Paradoxical growth has been reported as an adaptive mechanism to overcome the effects of caspofungin, although the consequence of this effect on the treatment of Candida infections is still unclear.14 Inhibition of fungal membrane efflux pumps possibly increases the susceptibility to antifungal drugs,30 mainly azole drugs, as observed with Candida spp.17,26,29 Thus, promethazine-induced efflux pump inhibition was evaluated using flow cytometry by measuring the efflux of R6G, which is a substrate of ATP-binding cassette (ABC) transporters, a class of membrane proteins that actively extrude azole drugs from the cells, conferring resistance.25 Only one of the two azole-resistant isolates showed significantly higher efflux of R6G in the presence of glucose than that obtained for glucose-deprived cells, showing a patent efflux activity. As for the effects of promethazine against this azole-resistant isolate, when cells were preincubated for 1 h with promethazine, an increase in intracellular fluorescence was observed for this azole-resistant strain with enhanced efflux activity, demonstrating that promethazine hindered the efflux of R6G. This decrease in efflux activity caused by promethazine may be due to disturbances in cell membrane function or alterations in ATP production/metabolism. Changes in cell size/granularity were observed when cells were exposed to promethazine, which are associated with cell death. This alteration may be associated with the possible action of promethazine on cell membrane, as indicated by the analysis of membrane integrity using propidium iodide, a membrane-impermeant DNA-intercalating dye that is used to detect increased permeability of cell membranes, after antifungal treatment.31 It is know that compounds that cause propidium iodide influx may directly affect fungal membranes, leading to fungal cell death.32,33 Alterations in membrane integrity and function could be another mechanism associated with the synergistic interaction between promethazine and azole antifungals. An interesting fact noticed is that, at higher concentrations of promethazine (256 μg/mL), a decrease in intracellular fluorescence is observed, indicating a decrease in plasma membrane damage. However, as observed in the time-kill curve assay, higher concentrations of promethazine start kill C. tropicalis cells after 2 h incubation. This fact indicates that membrane damage is not the major effect leading to death when C. tropicalis cells are exposed to high concentrations of promethazine. Moreover, the mitochondrial function was altered when C. tropicalis cells were treated with promethazine, suggesting the interference of this compound on mitochondrial respiratory function. Decrease in Rh123 fluorescence, a cationic dye used to label mitochondria in living cells, is related to depolarization and de-energization of mitochondria, which leads to reduction in mitochondrial function.34,35 It is important to highlight that collapse of mitochondrial transmembrane potential can cause release of pro-apoptotic factors into the cytosol, leading to cell death.27 Moreover, specific drugs that target fungal mitochondria could act in synergy with triazoles since this organelle acts as an energy conduit that is needed for many cell processes, including ergosterol biosynthesis.36 In fact, a mitochondrial respiratory dysfunction leads to a decrease in NADPH and oxygen, which are respectively required for squalene dimerization and the conversion of squalene into 2,3 oxidosqualene, previous steps to that targeted by azoles.37 Thus, the synergism between azoles and promethazine may also be associated with the action of the latter on fungal mitochondria and cell structure, leading to the decrease in efflux activity and ergosterol biosynthesis, resulting in the reduction of the antifungal MICs against azole-resistant strains. Alterations in mitochondrial function and cell morphology were observed in both azole-resistant and azole-susceptible strains. However, synergism between azoles and promethazine was only observed against azole-resistant isolates. This difference could be associated with differences in cell metabolism, as azole-resistant strains commonly present increased metabolic activity, often associated with increased respiration and gene overexpression, including ERG11, CDR and MDR.6 Additionally, Denerdi et al.38 reported that the combination of tacrolimus, a calcineurin inhibitor, also known for its efflux pump inhibiting properties,39 and azoles showed high percentages of synergy against fluconazole-resistant C. glabrata but not against susceptible strains. Moreover, antagonistic interactions between tacrolimus and fluconazole were also observed against susceptible strains, similar to the results found in this study, where antagonistic interactions between promethazine and azoles (fluconazole and voriconazole) against the susceptible strain were observed. Therefore, these results corroborate those found in our study, since no synergistic interactions between promethazine and azole drugs were observed. In summary, the present study demonstrated the synergistic effect between promethazine and azoles against resistant C. tropicalis isolates, reversing the resistance phenotype to all tested azoles. Promethazine presented an antibiofilm activity at a concentration 4 times the MIC against planktonic cells. Promethazine showed antifungal activity, altering cell size/granularity, membrane integrity and mitochondrial function, which are potential mechanisms of promethazine-mediated cell damage against C. tropicalis cells. Supplementary material Supplementary data are available at MMYCOL online. Funding This work was supported by the National Council for Scientific and Technological Development (CNPq; Brazil; Process number: 443167/2014-1). Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Negri M, Silva S, Henriques M, Oliveira R. Insights into Candida tropicalis nosocomial infections and virulence factors. Eur J Clin Microbiol Infect Dis . 2012; 31: 1399– 1412. Google Scholar CrossRef Search ADS PubMed  2. Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J. Candida glabrata, Candida parapsilosis and Candida tropicalis: biology, epidemiology, pathogenicity and antifungal resistance. FEMS Microbiol Rev . 2012; 362: 288– 305. Google Scholar CrossRef Search ADS   3. Da Costa KRC, Ferreira JC, Komesu MC, Candido RC. 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Abstract

Abstract The aim of this study was to evaluate the effect of promethazine on the antifungal minimum inhibitory concentrations against planktonic cells and mature biofilms of Candida tropicalis, as well as investigate its potential mechanisms of cell damage against this yeast species. Three C. tropicalis isolates (two azole-resistant and one azole-susceptible) were evaluated for their planktonic and biofilm susceptibility to promethazine alone and in combination with itraconazole, fluconazole, voriconazole, amphotericin B, and caspofungin. The antifungal activity of promethazine against C. tropicalis was investigated by performing time-kill curve assays and assessing rhodamine 6G efflux, cell size/granularity, membrane integrity, and mitochondrial transmembrane potential, through flow cytometry. Promethazine showed antifungal activity against planktonic cells and biofilms at concentrations of 64 and 128 μg/ml, respectively. The addition of two subinhibitory concentrations of promethazine reduced the antifungal MICs for all tested azole drugs against planktonic growth, reversing the resistance phenotype to all azoles. Promethazine decreased the efflux of rhodamine 6G in an azole-resistant strain. Moreover, promethazine decreased cell size/granularity and caused membrane damage, and mitochondrial membrane depolarization. In conclusion, promethazine presented synergy with azole antifungals against resistant C. tropicalis and exhibited in vitro cytotoxicity against C. tropicalis, altering cell size/granularity, membrane integrity, and mitochondrial function, demonstrating potential mechanisms of cell damage against this yeast species. Promethazine, Candida tropicalis, azole resistance, synergism, biofilm Introduction C. tropicalis is frequently isolated from patients with candidiasis, and it currently is the first or second most frequent non-Candida albicans Candida species involved in cases of candidemia and candiduria.1 Azole resistance in C. tropicalis has been frequently reported,2 especially among clinical isolates,3–5 including those showing cross-resistance to azole antifungals. Azole resistance in C. tropicalis results from the overexpression and/or mutation in the ERG11 gene that encodes the cytochrome P450 lanosterol 14α-demethylase,6 the primary target for azoles. Furthermore, at least two families of multidrug transporters are involved in the development of azole resistance in Candida spp.,7 due to the up-regulation of genes that encode the efflux proteins CDR1 and MDR1.8,9 In addition, alternative mechanisms, such as biofilm formation and mitochondrial defects, have also been reported as strategies to develop azole resistance in Candida spp.10,11 Candida biofilms are one of the main leading causes of infections related to medical devices.12 Biofilm sessile cells are less susceptible to antimicrobial drugs and host defenses, which complicates the treatment of the infection.13 In this context, C. tropicalis biofilms show reduced susceptibility to azole drugs.14 Thus, the search for compounds with antifungal and antibiofilm activities becomes relevant. Promethazine is a first-generation H1 receptor antagonist, with antihistamine and antiemetic effects, which belongs to phenothiazines,15 a group of compounds known to present efflux pump inhibitory properties.16 Some studies have evaluated the antifungal activity of promethazine;17,18 however, further investigations are needed to better understand these inhibitory effects on fungal cells. Thus, the aim of this study was to evaluate the effect of promethazine on the antifungal minimum inhibitory concentrations against planktonic cells and mature biofilms of C. tropicalis, as well as investigate its potential mechanisms of cell damage against this yeast species. Methods Isolates Three C. tropicalis strains, two azole-resistant and one azole-susceptible, from urine, bronchoalveolar lavage, and tracheal aspirate, respectively, were used in this study. These isolates belong to the Culture Collection of the Specialized Medical Mycology Center, Brazil, where they are stored in saline solution at 4°C. Strain identification was based on micromorphological features on cornmeal-Tween 80 agar, carbon and nitrogen assimilation, and carbon fermentation.14 Single colonies were transferred to potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA) for further assays. The phenotypical identification of the isolates was confirmed by matrix-assisted laser desorption-ionization time of flight (MALDI-TOF), as described by Lima-Neto et al.19 The equipment used was MALDI-TOF Autoflex III Mass Spectrometer (Bruker Daltonics Inc., Billerica, MA, USA/Germany), equipped with a Nd:YAG (neodymium-doped yttrium aluminum garnet; Nd:Y3Al5O12) laser of 1064 nm, set to a 66% power. The mass range from 2000 to 20,000 Da was recorded using a linear mode with a delay of 104 ns and an acceleration voltage of +20 kV. The resulting peak lists were exported to the software MALDI Biotyper™ 3.0 (Bruker Daltonics, Bremem, Germany), which provided the final identification of the isolates as C. tropicalis. Antifungal susceptibility testing Minimum inhibitory concentrations (MICs) for amphotericin B, itraconazole, fluconazole, voriconazole, caspofungin, and promethazine were investigated by broth microdilution, according to the document M27-A3.20 In order to determine the susceptibility of the planktonic cells, drugs were tested at concentrations ranging from 0.00390625 to 8 μg/ml for caspofungin, from 0.0625 to 16 μg/ml for amphotericin B, from 0.125 to 1024 for fluconazole, from 0.03125 to 512 μg/ml for itraconazole and voriconazole, and from 1 to 512 μg/ml for promethazine. The maximum or minimum concentration tested was adapted in order to determine the MICs. Serial twofold dilutions of each antifungal were prepared in RPMI 1640 medium with L-glutamine and without sodium bicarbonate (Sigma Chemical Co., St. Louis, MO, USA), buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS; Sigma Chemical Co., USA). Susceptibility testing for planktonic cells was performed in 96-well plates at 35°C for 24 hours. All isolates were tested in duplicate. For amphotericin B, the MIC was defined as the lowest drug concentration that inhibits 100% of fungal growth, while for azoles, caspofungin and promethazine, MICs were defined as the lowest drug concentration capable of inhibiting 50% of fungal growth, when compared to the drug-free growth control.20C. tropicalis ATCC 750 was used as a reference strain, which was kindly given to our research group by Coleção de Microrganismos de Referência em Vigilância Sanitária-CMRVS, FIOCRUZ-INCQS, Rio de Janeiro, RJ, Brazil, where it is maintained under the number INCQS 40281. C. parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were included as quality control for each test. Isolates with MICs ≥1.0 μg/ml, ≥1.0 μg/ml, ≥8.0 μg/ml, ≥1.0 μg/ml were considered resistant to itraconazole, voriconazole, fluconazole and caspofungin, respectively.20,21 Paradoxical growth after 120 h of incubation was confirmed, when a significant increase in cell growth was observed with caspofungin concentrations that were at least two concentrations above the MIC.22 Minimum fungicidal concentration (MFC) for promethazine was determined by seeding the content from all visually clear wells onto PDA plates. The MFC was the lowest drug concentration that killed 99.9% of the fungal inoculums.23 Time-kill curve study Time-kill curve assay was performed in standard RPMI medium, according to the method described by Cantón et al.,23 with modifications. Previously, the isolates were subcultured on PDA and grown at 35°C, for 24 h. The inoculum was adjusted to the density of 0.5 on McFarland's turbidity standard. The adjusted inoculum was diluted 1:20 in RPMI containing promethazine at 16, 32, 64, or 128μg/ml, with a final volume of 5 ml. This procedure yielded an initial inoculum of 4–6 × 104 cfu/ml. Then, the fungal suspensions were incubated at 35°C without agitation. At seven predetermined time points (0, 2, 4, 6, 12, 24, and 48 h), a 0.1-ml aliquot was removed from the drug-free control tube and each tube containing the test solution and serially diluted in sterile saline, reaching up to 6 dilutions, depending on the amount of fungal growth and drug concentration. Aliquots of 0.1 ml were spread onto PDA plates and incubated at 35°C for 24 to 48 h to determine the number of colony-forming unit per milliliter. Time-kill assays were conducted in duplicate. Biofilm susceptibility testing For the evaluation of antifungal and promethazine susceptibility of sessile cells, biofilms were prepared as described by Brilhante et al.14 Yeast suspensions were adjusted to reach 1 × 106 cells/mL in RPMI medium and then 200 μl were transferred to flat bottomed 96-well polystyrene plates. After 24 h of biofilm formation at 37°C, the medium was discarded and each well was washed three times with sterile phosphate-buffered saline (PBS). Afterward, 200 μl of RPMI 1640 medium containing serial twofold dilutions of each tested drug were added to the wells containing mature C. tropicalis biofilms and the plates were incubated at 37°C for further 48 hours. The drugs were tested at the following concentration ranges: 0.5–512 μg/ml for fluconazole; 0.125–128 μg/ml for itraconazole, voriconazole, amphotericin B; 0.0625–64 μg/ml for caspofungin and 4–2048 μg/ml for promethazine. For each tested strain, drug-free biofilm growth (growth control) and biofilm-free wells (sterility control) were included. Finally, biofilm metabolic activity was quantified using the XTT-reduction assay. Aliquots of 100 μl of XTT menadione solution [0.1 mg/ml XTT, 1 mM menadione (Sigma Chemical Co.)] were added to each well, and the plates were incubated in the dark for 2 h at 37°C, followed by spectrophotometric readings at 492 nm with a microtiter plate reader. Antifungal sessile minimum inhibitory concentrations (SMIC) against biofilm cells were determined as the concentration able of causing 50% (SMIC50) and 90% (SMIC90) inhibition of biofilm metabolic activity, when compared to the metabolic activity of the drug-free biofilm growth control. For caspofungin, the occurrence of paradoxical growth was considered when a metabolic activity of 20% or more of that obtained for the drug-free growth control was observed at drug concentrations above the SMIC.14 All assays were carried out in duplicate. Evaluation of drug interaction For the evaluation of the interaction between promethazine and the antifungal drugs, two sub-inhibitory concentrations of promethazine (16 and 8 μg/ml) were used in the analyses of each isolate. The antifungal susceptibility analysis of planktonic and sessile cells was performed, as previously described, with amphotericin B, caspofungin, fluconazole, itraconazole, and voriconazole. Planktonic and sessile cells were incubated in the presence of the combination of promethazine and antifungals and incubated as above described. C. tropicalis ATCC 750 was also included as reference control strain. Afterward, the fractional inhibitory concentration index (FICI) was calculated for each promethazine combination, in order to evaluate drug interaction, which was considered to be synergistic when FICI ≤ 0.5, indifferent when 0.5 < FICI ≤ 4, and antagonistic when FICI > 4.17,24 Confocal laser scanning microscopy (CLSM) Biofilms of C. tropicalis were analyzed by CLSM, according to Brilhante et al.14 Biofilms were formed directly on ThermanoxTM coverslips, as described above, and different promethazine concentrations (32, 64, 128, 256, 512, 1024, and 2048 μg/ml) were added to the samples. Biofilms were incubated for additional 48 h at 37°C. Control (unexposed) samples were incubated in RPMI 1640 only. After incubation, the ThermanoxTM coverslips containing the adhered biofilm was transferred to a glass bottom Petri dish, where Live/Dead (Invitrogen TM151) fluorescent dye was added to cover the surface of the ThermanoxTM coverslips. Afterward, the coverslips were evaluated under a Confocal Microscope (Nikon C2), at 488 nm for the detection of the SYTO 9 fluorescent dye (live cells) and at 561 nm for the detection of the propidium iodide (dead cells).14 For image analyses, five equidistant points of the coverlips were submitted to Z-stacked capture of the images, for three-dimensional image. Scanning electron microscopy (SEM) Biofilms of C. tropicalis were analyzed by SEM, as described in the previous section (CLSM). After incubation, biofilm supernatants were removed, and slides were washed with cacodylate buffer (0.15 M) twice. Biofilms were then covered with glutaraldehyde (2.5% in cacodylate buffer) and incubated at 4°C overnight. After incubation, biofilms were washed twice with cacodylate buffer, and slides were dehydrated in ascending ethanol concentrations (50, 70, 80, 95, and 100%) twice, for 10 minutes each concentration. Slides were dried at room temperature and covered with hexamethyldisilazane for 30 minutes. Slides were dried overnight, coated with 10 nm of gold (Emitech Q150T), and observed with a FEI Inspect S50 scanning electron microscope, in the high vacuum mode at 15 kV, and images were processed with Photoshop (Adobe Systems, San Jose, CA, USA) software.14 Analysis of efflux pump activity by flow cytometry The activity of efflux pumps was evaluated through flow cytometry by measuring the efflux of rhodamine 6G (R6G), as described by Jiang et al.6 and Ivnitski-Steele et al.,25 with modifications. Yeast cells (106) in YEPD broth (1% yeast extract, 2% peptone, 2% dextrose) were suspended in 2 ml of PBS and incubated for 4 h at 30°C with constant shaking to induce starvation. Starved cells were pre-incubated with promethazine at 8, 16, and 32 μg/ml, for 1 h. After incubation, 15 μM R6G (7.5 mM stock in DMSO) were added and incubated at 30°C, for 20 min, in the dark. Then cells were washed with cold PBS, followed by the addition of glucose (20 mM in PBS) and incubation at 30°C for 30 min. Afterward, cells were washed twice with cold sterile PBS. The fluorescence of each condition was immediately quantified using a FACSCalibur flow cytometer (BD Biosciences), with measurement of 10,000 cells per sample. Each assay was performed in triplicate and an azole-resistant C. parapsilosis, which is known to have an enhanced efflux activity,26 was used as a positive control. Cellular debris was omitted from the analysis. Determination of cell size/granulariry and membrane integrity Cell size and granularity and membrane integrity of the fungal cells were evaluated by flow cytometry. Fungal strains were exposed to promethazine (64, 128, and 256 μg/ml), amphotericin B (control drug, 2 μg/ml), and fluconazole (control drug, 64 μg/ml) in RPMI medium at a final concentration of 106 cells/ml at 35°C, for 2 and 4 h. Then, the cells were washed with PBS and incubated with 2 μg/ml propidium iodide (PI), in the dark, for 30 min. The cellular fluorescence was then determined using flow cytometry, in a FACSCalibur flow cytometer. Each assay was performed in triplicate. A total of 10,000 cells were evaluated per assay, with the cellular debris omitted from the analysis.27 Measurement of mitochondrial transmembrane potential (Δψm) The mitochondrial transmembrane potential was determined by the retention of rhodamine 123 (Rho123) dye by fungal cells, after exposure to promethazine (64, 128, and 256 μg/ml), amphotericin B (control drug, 2 μg/ml), and fluconazole (control drug, 64 μg/ml) in RPMI medium at a final concentration of 106 cells/ml at 35°C, for 2 and 4 h. After exposure, the cells were washed with PBS incubated with 5 μg/ml Rho123, in the dark, at 37°C for 30 min, and then washed twice with PBS. Their fluorescence was measured using flow cytometry, in a FACSCalibur flow cytometer. Each assay was performed in triplicate. A total of 10,000 cells were evaluated per experiment, and cellular debris was omitted from the analysis.27 Statistical analysis All tests were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. P-values < .05 were considered statistically significant. The statistical analyses were performed with GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). Results Results regarding antifungal susceptibility tests are shown in Table 1. Promethazine MIC and MFC were 64 μg/ml and 256 μg/ml, respectively. Fluconazole MICs against azole-resistant isolates were 256 and 512 μg/ml, voriconazole MICs were 32 and 64 μg/ml, and those for itraconazole were 128 μg/ml. Antifungal MICs against the azole-susceptible strain were 0.5, 0.0625, and 0.0625 μg/ml for fluconazole, itraconazole, and voriconazole, respectively. Amphotericin B and caspofungin MIC ranges were 0.25–1 μg/ml and 0.0078–0.0156 μg/ml, respectively. Table 1. Minimum inhibitory concentrations of promethazine and antifungal drugs isolated and in combination with promethazine against Candida tropicalis isolates.   MIC (μg/ml)  FICI    Azole-resistant      Azole-resistant        strains      strains            Azole-susceptible  ATCC 750      Azole-susceptible  ATCC 750  Drugs  Strain 1  Strain 2  strain    Strain 1  Strain 2  strain    PRM  64  64  64            256 (MFC)  256 (MFC)  256 (MFC)  -  -  -  AMB  0.5  1  0.25  0.25  -  -  -  -  AMB+PRM 8  0.125  0.125  0.0625  0.0625  0.375S  0.250S  0.325S  0.325S  AMB+PRM 16  0.125  0.0625  0.0625  0.0625  0.5S  0.312S  0.5S  0.5S  CAS*  0.0156  0.0078  0.0078  0.03125  -  -  -  -  CAS+PRM 8  0.0078  0.0039  0.0078  0.03125  0.625I  0.625I  1.125I  1.125I  CAS+PRM 16  0.0078  0.0039  0.0039  0.0156  0.75I  0.75I  0.75I  0.75I  FLC  512  256  0.5  0.5  -  -  -  -  FLC+PRM 8  4  4  4  4  0.133S  0.141S  8.125A  8.125A  FLC+PRM 16  4  4  2  4  0.258S  0.266S  4.25A  8.25A  ITC  128  128  0.0625  0.0625  -  -  -  -  ITC+PRM 8  0.0625  0.125  0.125  0.03125  0.125S  0.126S  2.125I  0.625I  ITC+PRM 16  0.03125  0.0625  0.03125  0.0156  0.25S  0.25S  0.75I  0.5S  VRC  32  64  0.0625  0.03125  -  -  -  -  VRC+PRM 8  0.5  1  0.25  0.25  0.141S  0.141S  4.125A  2.125I  VRC+PRM 16  0.25  0.5  0.25  0.125  0.258S  0.258S  4.25A  4.25A    MIC (μg/ml)  FICI    Azole-resistant      Azole-resistant        strains      strains            Azole-susceptible  ATCC 750      Azole-susceptible  ATCC 750  Drugs  Strain 1  Strain 2  strain    Strain 1  Strain 2  strain    PRM  64  64  64            256 (MFC)  256 (MFC)  256 (MFC)  -  -  -  AMB  0.5  1  0.25  0.25  -  -  -  -  AMB+PRM 8  0.125  0.125  0.0625  0.0625  0.375S  0.250S  0.325S  0.325S  AMB+PRM 16  0.125  0.0625  0.0625  0.0625  0.5S  0.312S  0.5S  0.5S  CAS*  0.0156  0.0078  0.0078  0.03125  -  -  -  -  CAS+PRM 8  0.0078  0.0039  0.0078  0.03125  0.625I  0.625I  1.125I  1.125I  CAS+PRM 16  0.0078  0.0039  0.0039  0.0156  0.75I  0.75I  0.75I  0.75I  FLC  512  256  0.5  0.5  -  -  -  -  FLC+PRM 8  4  4  4  4  0.133S  0.141S  8.125A  8.125A  FLC+PRM 16  4  4  2  4  0.258S  0.266S  4.25A  8.25A  ITC  128  128  0.0625  0.0625  -  -  -  -  ITC+PRM 8  0.0625  0.125  0.125  0.03125  0.125S  0.126S  2.125I  0.625I  ITC+PRM 16  0.03125  0.0625  0.03125  0.0156  0.25S  0.25S  0.75I  0.5S  VRC  32  64  0.0625  0.03125  -  -  -  -  VRC+PRM 8  0.5  1  0.25  0.25  0.141S  0.141S  4.125A  2.125I  VRC+PRM 16  0.25  0.5  0.25  0.125  0.258S  0.258S  4.25A  4.25A  Strain 1: azole-resistant strain with enhanced efflux activity. Strain 2: azole-resistant strain with decreased efflux activity. AMB, amphotericin B; CAS, caspofungin; FICI, fractional inhibitory concentration index; FLC, fluconazole; ITC, itraconazole; MFC, minimum fungicidal concentration; MIC, minimum inhibitory concentration; PRM, promethazine; PRM 8, promethazine at 8 μg/ml; PRM 16, promethazine at 16 μg/ml; VRC, voriconazole. Ssynergistic interaction (FICI ≤ 0.5); I indifferent interaction (0.5 > FICI ≤ 4); A antagonistic interaction (FICI > 4). *Paradoxical growth effect of caspofungin alone was observed at 8 μg/ml only against azole susceptible isolate, and at 4 to 16 μg/ml when combined with promethazine (8 and 16 μg/ml) against azole susceptible and resistant C. tropicalis isolates. View Large Table 1. Minimum inhibitory concentrations of promethazine and antifungal drugs isolated and in combination with promethazine against Candida tropicalis isolates.   MIC (μg/ml)  FICI    Azole-resistant      Azole-resistant        strains      strains            Azole-susceptible  ATCC 750      Azole-susceptible  ATCC 750  Drugs  Strain 1  Strain 2  strain    Strain 1  Strain 2  strain    PRM  64  64  64            256 (MFC)  256 (MFC)  256 (MFC)  -  -  -  AMB  0.5  1  0.25  0.25  -  -  -  -  AMB+PRM 8  0.125  0.125  0.0625  0.0625  0.375S  0.250S  0.325S  0.325S  AMB+PRM 16  0.125  0.0625  0.0625  0.0625  0.5S  0.312S  0.5S  0.5S  CAS*  0.0156  0.0078  0.0078  0.03125  -  -  -  -  CAS+PRM 8  0.0078  0.0039  0.0078  0.03125  0.625I  0.625I  1.125I  1.125I  CAS+PRM 16  0.0078  0.0039  0.0039  0.0156  0.75I  0.75I  0.75I  0.75I  FLC  512  256  0.5  0.5  -  -  -  -  FLC+PRM 8  4  4  4  4  0.133S  0.141S  8.125A  8.125A  FLC+PRM 16  4  4  2  4  0.258S  0.266S  4.25A  8.25A  ITC  128  128  0.0625  0.0625  -  -  -  -  ITC+PRM 8  0.0625  0.125  0.125  0.03125  0.125S  0.126S  2.125I  0.625I  ITC+PRM 16  0.03125  0.0625  0.03125  0.0156  0.25S  0.25S  0.75I  0.5S  VRC  32  64  0.0625  0.03125  -  -  -  -  VRC+PRM 8  0.5  1  0.25  0.25  0.141S  0.141S  4.125A  2.125I  VRC+PRM 16  0.25  0.5  0.25  0.125  0.258S  0.258S  4.25A  4.25A    MIC (μg/ml)  FICI    Azole-resistant      Azole-resistant        strains      strains            Azole-susceptible  ATCC 750      Azole-susceptible  ATCC 750  Drugs  Strain 1  Strain 2  strain    Strain 1  Strain 2  strain    PRM  64  64  64            256 (MFC)  256 (MFC)  256 (MFC)  -  -  -  AMB  0.5  1  0.25  0.25  -  -  -  -  AMB+PRM 8  0.125  0.125  0.0625  0.0625  0.375S  0.250S  0.325S  0.325S  AMB+PRM 16  0.125  0.0625  0.0625  0.0625  0.5S  0.312S  0.5S  0.5S  CAS*  0.0156  0.0078  0.0078  0.03125  -  -  -  -  CAS+PRM 8  0.0078  0.0039  0.0078  0.03125  0.625I  0.625I  1.125I  1.125I  CAS+PRM 16  0.0078  0.0039  0.0039  0.0156  0.75I  0.75I  0.75I  0.75I  FLC  512  256  0.5  0.5  -  -  -  -  FLC+PRM 8  4  4  4  4  0.133S  0.141S  8.125A  8.125A  FLC+PRM 16  4  4  2  4  0.258S  0.266S  4.25A  8.25A  ITC  128  128  0.0625  0.0625  -  -  -  -  ITC+PRM 8  0.0625  0.125  0.125  0.03125  0.125S  0.126S  2.125I  0.625I  ITC+PRM 16  0.03125  0.0625  0.03125  0.0156  0.25S  0.25S  0.75I  0.5S  VRC  32  64  0.0625  0.03125  -  -  -  -  VRC+PRM 8  0.5  1  0.25  0.25  0.141S  0.141S  4.125A  2.125I  VRC+PRM 16  0.25  0.5  0.25  0.125  0.258S  0.258S  4.25A  4.25A  Strain 1: azole-resistant strain with enhanced efflux activity. Strain 2: azole-resistant strain with decreased efflux activity. AMB, amphotericin B; CAS, caspofungin; FICI, fractional inhibitory concentration index; FLC, fluconazole; ITC, itraconazole; MFC, minimum fungicidal concentration; MIC, minimum inhibitory concentration; PRM, promethazine; PRM 8, promethazine at 8 μg/ml; PRM 16, promethazine at 16 μg/ml; VRC, voriconazole. Ssynergistic interaction (FICI ≤ 0.5); I indifferent interaction (0.5 > FICI ≤ 4); A antagonistic interaction (FICI > 4). *Paradoxical growth effect of caspofungin alone was observed at 8 μg/ml only against azole susceptible isolate, and at 4 to 16 μg/ml when combined with promethazine (8 and 16 μg/ml) against azole susceptible and resistant C. tropicalis isolates. View Large The tested subinhibitory concentrations of promethazine (8 and 16 μg/ml) reduced the antifungal MICs and reversed the azole resistance phenotype of the two azole-resistant isolates. The MICs against the azole-resistant isolates reduced 64- to 128-fold for fluconazole and voriconazole and 1024- to 4096-fold for itraconazole (Table 1). However, no synergistic interaction was observed for the combination of promethazine and azoles against the azole-susceptible strain and the reference strain (C. tropicalis ATCC 750), and antagonism was observed when promethazine was combined with fluconazole and voriconazole. Promethazine reduced the MICs for amphotericin B and caspofungin two- to 16-fold against the three C. tropicalis isolates and the reference strain. Paradoxical growth effect of caspofungin alone was only observed against the azole susceptible isolate, at 8 μg/ml. As for the combination of caspofungin and subinhibitory concentrations of promethazine (8 and 16 μg/ml), paradoxical growth was observed at 4 to 16 μg/ml of caspofungin against azole susceptible and resistant isolates (Table 1). The killing activity of promethazine against C. tropicalis is shown in Figure 1. Reduction in the number of cfu per milliliter was observed after 2 h of incubation of the yeast cells at different concentrations of the drug. At concentrations of 64, 128, and 256 μg/ml, the time required to kill 50% of the initial inoculum was 2 h. At the concentration four times the MIC (256 μg/ml), 99% reduction of live fungal cells was observed after 4 h of incubation, and no viable cells were observed after 12 h of incubation. Figure 1. View largeDownload slide Representative plots of mean ± standard deviation of colony-forming unit counts for the establishment of a time-kill curve for Candida tropicalis after exposure to promethazine at 32 μg/ml (square), 64 μg/ml (diamond), 128 μg/ml (circle), and 256 μg/ml (×). Drug-free growth control was also added for comparative purposes (triangle). Figure 1. View largeDownload slide Representative plots of mean ± standard deviation of colony-forming unit counts for the establishment of a time-kill curve for Candida tropicalis after exposure to promethazine at 32 μg/ml (square), 64 μg/ml (diamond), 128 μg/ml (circle), and 256 μg/ml (×). Drug-free growth control was also added for comparative purposes (triangle). Regarding mature biofilm susceptibility, promethazine showed SMIC50 of 128 μg/ml and SMIC90 of 256 μg/ml, 2 times and 4 times the MIC for planktonic cells, respectively (Table 2). Antifungal SMIC50 and SMIC90 against C. tropicalis mature biofilm were 8 and 32 μg/ml for amphotericin B, <0.0625 μg/ml for caspofungin, >512 μg/ml for fluconazole and >128 μg/ml for itraconazole and voriconazole. Promethazine did not significantly modify the biofilm susceptibility to azoles, amphotericin B and caspofungin (Table 2). The minimum caspofungin concentration that induced paradoxical biofilm growth decreased four- to eightfold in the presence of promethazine (Table 2). Table 2. Sessile minimum inhibitory concentrations of promethazine and antifungal drugs isolated and in combination with promethazine against Candida tropicalis mature biofilms Drugs  SMIC50  SMIC90    (μg/ml)  (μg/ml)  PRM  128  256  AMB  8  32  AMB+PRM 8  16  64  AMB+PRM 16  16  64  CAS*  <0.0625  <0.0625  CAS+PRM 8*  <0.0625  <0.0625  CAS+PRM16*  <0.0625  <0.0625  FLC  >512  >512  FLC+PRM 8  >512  >512  FLC+PRM 16  >512  >512  ITC  >128  >128  ITC+PRM 8  >128  >128  ITC+PRM 16  >128  >128  VRC  >128  >128  VRC+PRM 8  >128  >128  VRC+PRM 16  >128  >128  Drugs  SMIC50  SMIC90    (μg/ml)  (μg/ml)  PRM  128  256  AMB  8  32  AMB+PRM 8  16  64  AMB+PRM 16  16  64  CAS*  <0.0625  <0.0625  CAS+PRM 8*  <0.0625  <0.0625  CAS+PRM16*  <0.0625  <0.0625  FLC  >512  >512  FLC+PRM 8  >512  >512  FLC+PRM 16  >512  >512  ITC  >128  >128  ITC+PRM 8  >128  >128  ITC+PRM 16  >128  >128  VRC  >128  >128  VRC+PRM 8  >128  >128  VRC+PRM 16  >128  >128  SMIC50: sessile minimum inhibitory concentration that causes 50% inhibition of biofilm metabolic activity, measured through XTT reduction assay; SMIC90: sessile minimum inhibitory concentration that causes 90% inhibition of biofilm metabolic activity, measured through XTT reduction assay. AMB, amphotericin B; CAS, caspofungin; FLC, fluconazole; ITC, itraconazole; PRM, promethazine; PRM 8, promethazine at 8 μg/ml; PRM 16, promethazine at 16 μg/ml; VRC: voriconazole. *Paradoxical growth effect of caspofungin alone was observed at 4 μg/ml, and at 1 and 0.5 μg/ml, when combined with promethazine at 8 and 16 μg/ml, respectively. View Large Table 2. Sessile minimum inhibitory concentrations of promethazine and antifungal drugs isolated and in combination with promethazine against Candida tropicalis mature biofilms Drugs  SMIC50  SMIC90    (μg/ml)  (μg/ml)  PRM  128  256  AMB  8  32  AMB+PRM 8  16  64  AMB+PRM 16  16  64  CAS*  <0.0625  <0.0625  CAS+PRM 8*  <0.0625  <0.0625  CAS+PRM16*  <0.0625  <0.0625  FLC  >512  >512  FLC+PRM 8  >512  >512  FLC+PRM 16  >512  >512  ITC  >128  >128  ITC+PRM 8  >128  >128  ITC+PRM 16  >128  >128  VRC  >128  >128  VRC+PRM 8  >128  >128  VRC+PRM 16  >128  >128  Drugs  SMIC50  SMIC90    (μg/ml)  (μg/ml)  PRM  128  256  AMB  8  32  AMB+PRM 8  16  64  AMB+PRM 16  16  64  CAS*  <0.0625  <0.0625  CAS+PRM 8*  <0.0625  <0.0625  CAS+PRM16*  <0.0625  <0.0625  FLC  >512  >512  FLC+PRM 8  >512  >512  FLC+PRM 16  >512  >512  ITC  >128  >128  ITC+PRM 8  >128  >128  ITC+PRM 16  >128  >128  VRC  >128  >128  VRC+PRM 8  >128  >128  VRC+PRM 16  >128  >128  SMIC50: sessile minimum inhibitory concentration that causes 50% inhibition of biofilm metabolic activity, measured through XTT reduction assay; SMIC90: sessile minimum inhibitory concentration that causes 90% inhibition of biofilm metabolic activity, measured through XTT reduction assay. AMB, amphotericin B; CAS, caspofungin; FLC, fluconazole; ITC, itraconazole; PRM, promethazine; PRM 8, promethazine at 8 μg/ml; PRM 16, promethazine at 16 μg/ml; VRC: voriconazole. *Paradoxical growth effect of caspofungin alone was observed at 4 μg/ml, and at 1 and 0.5 μg/ml, when combined with promethazine at 8 and 16 μg/ml, respectively. View Large Alterations in biofilm structure and morphological changes in biofilm-associated cells after exposure to different promethazine concentrations were observed using CLSM (Fig. 2) and SEM (Fig. 3). Fragmentation and reduction of viable cells and biomass were observed when biofilms were exposed to concentrations starting at 32 μg/ml of promethazine (MIC/2 against planktonic cells). Decrease in biofilm thickness was observed in all tested concentrations (Fig. 2). Biomass increased at higher concentrations (1024 and 2048 μg/ml, Fig. 2 and 3); however, no live cells were observed (Fig. 2). In addition, morphological changes in yeast cells were observed at 256 and 512 μg/ml, showing collapsed cells with wrinkled surface (Fig. 3). Figure 2. View largeDownload slide Confocal laser scanning microscopy images of Candida tropicalis mature biofilms after 48 h of incubation at different concentrations of promethazine (PRM). Notice the decrease in biofilm thickness and in the number of live cells at all tested concentrations, when compared to growth control. An increase in biomass and biofilm thickness is observed at the highest concentrations, however no live cells are detected. Magnification: 400 ×. Lasers: 488 nm (images a–d, SYTO9, viable cells), 561 nm (images e–h, propidium iodide, dead or damaged cells). Scale: 200 μm. Figure 2. View largeDownload slide Confocal laser scanning microscopy images of Candida tropicalis mature biofilms after 48 h of incubation at different concentrations of promethazine (PRM). Notice the decrease in biofilm thickness and in the number of live cells at all tested concentrations, when compared to growth control. An increase in biomass and biofilm thickness is observed at the highest concentrations, however no live cells are detected. Magnification: 400 ×. Lasers: 488 nm (images a–d, SYTO9, viable cells), 561 nm (images e–h, propidium iodide, dead or damaged cells). Scale: 200 μm. Figure 3. View largeDownload slide Scanning electron microscopy of Candida tropicalis mature biofilm after 48 h of incubation at different concentrations of promethazine (PRM). (a) drug-free growth control; (b) PRM 64 μg/ml, notice a reduced number of cells and loss of biofilm structure; (c) PRM 256 μg/ml, notice the increased number of morphologically altered cells (cells with empty appearance, arrows) and disrupted biofilm; (d) PRM 2048 μg/ml; notice an increase in biomass and presence of matrix remnants, with microscopically altered cells (wrinkled cells, arrows). Magnification: 5000 ×. Scale: 20 μm. Figure 3. View largeDownload slide Scanning electron microscopy of Candida tropicalis mature biofilm after 48 h of incubation at different concentrations of promethazine (PRM). (a) drug-free growth control; (b) PRM 64 μg/ml, notice a reduced number of cells and loss of biofilm structure; (c) PRM 256 μg/ml, notice the increased number of morphologically altered cells (cells with empty appearance, arrows) and disrupted biofilm; (d) PRM 2048 μg/ml; notice an increase in biomass and presence of matrix remnants, with microscopically altered cells (wrinkled cells, arrows). Magnification: 5000 ×. Scale: 20 μm. The uptake and efflux of R6G were quantified using flow cytometry to evaluate the activity of yeast efflux pumps and the effect of promethazine on this mechanism of survival and drug resistance. Initially, the efflux activity of the strains was assessed and one azole-resistant isolate showed significant decrease in intracellular fluorescence in the presence of glucose, when compared to the cells that were submitted to starvation (P < .05), demonstrating the efflux activity of this strain. Control strain (C. parapsilosis with enhanced efflux activity) also showed significant decrease in intracellular fluorescence in the presence of glucose in comparison with cells in starvation (P < .05). Afterward, the effect of promethazine was evaluated. Preincubation with promethazine (8, 16, and 32 μg/ml) induced an increase in the mean intracellular fluorescence intensity of the azole-resistant isolate that showed efflux activity, when compared to the unexposed control in the presence of glucose (P < .05). However, for the azole-resistant strain that did not show efflux activity and for the azole-susceptible strain, preincubation with promethazine caused a little but significant decrease in the mean fluorescence intensity of the cells, when compared to the cells in starvation and the unexposed control in the presence of glucose (P < .05). Promethazine also induced a slight but significant increase in the mean intracellular fluorescence intensity of the control strain (P < .05). A decrease in cell size/granularity was observed after 2 and 4 h incubation of C. tropicalis strains with promethazine at MIC, 2 × MIC, and 4 × MIC, similar to what was observed with amphotericin B (Fig. 4). As shown in Figure 5, a significant increase in the number of cells with plasma membrane damage was observed when yeast cells were exposed to different concentrations of promethazine, after 2 and 4 h of incubation, as demonstrated by the propidium iodide assay. Moreover, significant changes in the mitochondrial transmembrane potential were observed after 2 and 4 h of exposure to promethazine (Table 3). Rh123 fluorescence was lower in yeast cells exposed to promethazine, when compared to unexposed cells (P < .05). Decrease in Rh123 fluorescence was also observed when cells were exposed to amphotericin B. Figure 4. View largeDownload slide Representative flow cytometry analysis of changes in size (forward scatter, FSC) and granularity (side scatter, SSC) obtained for the azole-resistant (n = 2) and azole-susceptible (n = 1) strains of Candida tropicalis, after 4 h of exposure to promethazine (PRM), fluconazole (FLC) and amphotericin B (AMB). (a) untreated control; (b) FLC 64 μg/ml; (c) AMB 2 μg/ml; (d) PRM 64 μg/ml; (e) PRM 128 μg/ml; (f) PRM 256 μg/ml. Results are shown in mean fluorescence intensity of cells. Dashed lines represent cellular fluorescence of the untreated control (a). Reduction in cellular fluorescence indicates reduction in cells size (FSC) and/or granularity (SSC). Notice a reduction in cell size and granularity when cells are exposed to PRM and AMB, which is common for dying cells. Figure 4. View largeDownload slide Representative flow cytometry analysis of changes in size (forward scatter, FSC) and granularity (side scatter, SSC) obtained for the azole-resistant (n = 2) and azole-susceptible (n = 1) strains of Candida tropicalis, after 4 h of exposure to promethazine (PRM), fluconazole (FLC) and amphotericin B (AMB). (a) untreated control; (b) FLC 64 μg/ml; (c) AMB 2 μg/ml; (d) PRM 64 μg/ml; (e) PRM 128 μg/ml; (f) PRM 256 μg/ml. Results are shown in mean fluorescence intensity of cells. Dashed lines represent cellular fluorescence of the untreated control (a). Reduction in cellular fluorescence indicates reduction in cells size (FSC) and/or granularity (SSC). Notice a reduction in cell size and granularity when cells are exposed to PRM and AMB, which is common for dying cells. Figure 5. View largeDownload slide Representative analysis of mean fluorescence intensity of propidium iodide obtained for the azole-resistant (n = 2) and azole-susceptible (n = 1) strains of Candida tropicalis, following exposure to promethazine after 2 (a) and 4h (b) of incubation. Control: unexposed cells. AMB, amphotericin B. FLC, fluconazole. PRM, promethazine. Increase in intracellular fluorescence indicates plasma membrane damage. *indicates significant differences when compared to the unexposed control. Each bar represents the mean ± SD of triplicate readings of one representative strain. Figure 5. View largeDownload slide Representative analysis of mean fluorescence intensity of propidium iodide obtained for the azole-resistant (n = 2) and azole-susceptible (n = 1) strains of Candida tropicalis, following exposure to promethazine after 2 (a) and 4h (b) of incubation. Control: unexposed cells. AMB, amphotericin B. FLC, fluconazole. PRM, promethazine. Increase in intracellular fluorescence indicates plasma membrane damage. *indicates significant differences when compared to the unexposed control. Each bar represents the mean ± SD of triplicate readings of one representative strain. Table 3. Mean fluorescence intensity of rhodamine 123 (Rh123) in azole-resistant (n = 2) and azole-susceptible (n = 1) Candida tropicalis strains following exposure to promethazine.         PRM  Time of incubation  Control  FLC  AMB  64 μg/ml  128 μg/ml  256 μg/ml  2 h  72.85 ± 20.11  705.2 ± 202.1*  10.38 ± 2.01*  18.33 ± 5.28*  10.99 ± 2.56*  8.9 ± 0.59*  4 h  72.93 ± 14.71  1543 ± 11.86*  12.5 ± 1.18*  33 ± 6.79*  11.32 ± 0.86*  20.57 ± 9.67*          PRM  Time of incubation  Control  FLC  AMB  64 μg/ml  128 μg/ml  256 μg/ml  2 h  72.85 ± 20.11  705.2 ± 202.1*  10.38 ± 2.01*  18.33 ± 5.28*  10.99 ± 2.56*  8.9 ± 0.59*  4 h  72.93 ± 14.71  1543 ± 11.86*  12.5 ± 1.18*  33 ± 6.79*  11.32 ± 0.86*  20.57 ± 9.67*  Data represent mean fluorescence intensity. Control: untreated cells. AMB, amphotericin B 2 μg/ml. FLC, fluconazole 64 μg/ml. PRM, promethazine. Decrease in Rh123 fluorescence indicates depolarization and de-energization of mitochondria and impaired mitochondrial function. *significant difference when compared to untreated control. Data are expressed as mean ± SD of triplicate readings per tested strain. View Large Table 3. Mean fluorescence intensity of rhodamine 123 (Rh123) in azole-resistant (n = 2) and azole-susceptible (n = 1) Candida tropicalis strains following exposure to promethazine.         PRM  Time of incubation  Control  FLC  AMB  64 μg/ml  128 μg/ml  256 μg/ml  2 h  72.85 ± 20.11  705.2 ± 202.1*  10.38 ± 2.01*  18.33 ± 5.28*  10.99 ± 2.56*  8.9 ± 0.59*  4 h  72.93 ± 14.71  1543 ± 11.86*  12.5 ± 1.18*  33 ± 6.79*  11.32 ± 0.86*  20.57 ± 9.67*          PRM  Time of incubation  Control  FLC  AMB  64 μg/ml  128 μg/ml  256 μg/ml  2 h  72.85 ± 20.11  705.2 ± 202.1*  10.38 ± 2.01*  18.33 ± 5.28*  10.99 ± 2.56*  8.9 ± 0.59*  4 h  72.93 ± 14.71  1543 ± 11.86*  12.5 ± 1.18*  33 ± 6.79*  11.32 ± 0.86*  20.57 ± 9.67*  Data represent mean fluorescence intensity. Control: untreated cells. AMB, amphotericin B 2 μg/ml. FLC, fluconazole 64 μg/ml. PRM, promethazine. Decrease in Rh123 fluorescence indicates depolarization and de-energization of mitochondria and impaired mitochondrial function. *significant difference when compared to untreated control. Data are expressed as mean ± SD of triplicate readings per tested strain. View Large Discussion Azole resistance in C. tropicalis has become more common in the past years;4,5,28 therefore, the quest for alternatives to overcome this obstacle has become relevant. In the present study, promethazine, a compound derived from phenothiazine, was evaluated against planktonic and sessile cells of azole-resistant C. tropicalis. The azole-resistance phenotype was altered when C. tropicalis isolates were treated with subinhibitory concentrations of promethazine, with azole MIC reductions reaching up to 2048-fold. Similar results were observed by Castelo-Branco et al.17 and Brilhante et al.29 who tested promethazine in combination with azole antifungals against azole-resistant Candida species, showing synergistic interactions. The authors suggested that this synergism was due to efflux pump inhibition by promethazine, an important mechanism of resistance in Candida species. Iatta et al.30 suggested that the synergistic effect of promethazine and azoles against Malassezia species might be related to an increased efflux pump activity. This also seems to be the mechanism of drug interaction against C. tropicalis strains, since synergistic interactions between azoles and promethazine were only observed with azole-resistant isolates. Also, MIC reductions for amphotericin B and caspofungin were significant lower than those observed for azoles. Thus, possible mechanisms of cell damage caused by promethazine were assessed in order to better understand the interaction between promethazine and azole antifungals. The time-kill assay demonstrated that cell death is observed as soon as 2 h after incubation with promethazine at MIC, 2 × MIC and 4 × MIC. Cell death rate increases as drug concentration increases, reaching 100% death at 4 times the MIC (256 μg/ml), after 12 h of incubation, in agreement with the MFC results. This result indicates a possible fungicidal mechanism of promethazine against C. tropicalis.23 The mechanism of cell death of promethazine against C. tropicalis may be partially caused by damaged mitochondrial function, altered cell respiration and changes in cell structure, mainly membrane damage. Promethazine inhibited mature biofilms at concentrations twice the MIC for planktonic cells, showing an effective antibiofilm activity. Promethazine also promoted significant alterations in biofilm structure, decreasing the amount of hyphae and reducing its thickness. This structural change was more noticeable at lower concentrations of promethazine, while an increase in biomass was observed at the highest tested concentrations, although, live cells were not observed. The antibiofilm effects of promethazine alone may be of particular therapeutic interest, since biofilms are commonly associated with Candida infections and are inherently difficult to treat.12 On the other hand, promethazine did not reduce the antifungal inhibitory concentrations against C. tropicalis mature biofilms, contrasting the results obtained against planktonic cells. Moreover, paradoxical growth effect of caspofungin on planktonic and sessile cells was detected at lower caspofungin concentration, when this antifungal was combined with promethazine. Paradoxical growth has been reported as an adaptive mechanism to overcome the effects of caspofungin, although the consequence of this effect on the treatment of Candida infections is still unclear.14 Inhibition of fungal membrane efflux pumps possibly increases the susceptibility to antifungal drugs,30 mainly azole drugs, as observed with Candida spp.17,26,29 Thus, promethazine-induced efflux pump inhibition was evaluated using flow cytometry by measuring the efflux of R6G, which is a substrate of ATP-binding cassette (ABC) transporters, a class of membrane proteins that actively extrude azole drugs from the cells, conferring resistance.25 Only one of the two azole-resistant isolates showed significantly higher efflux of R6G in the presence of glucose than that obtained for glucose-deprived cells, showing a patent efflux activity. As for the effects of promethazine against this azole-resistant isolate, when cells were preincubated for 1 h with promethazine, an increase in intracellular fluorescence was observed for this azole-resistant strain with enhanced efflux activity, demonstrating that promethazine hindered the efflux of R6G. This decrease in efflux activity caused by promethazine may be due to disturbances in cell membrane function or alterations in ATP production/metabolism. Changes in cell size/granularity were observed when cells were exposed to promethazine, which are associated with cell death. This alteration may be associated with the possible action of promethazine on cell membrane, as indicated by the analysis of membrane integrity using propidium iodide, a membrane-impermeant DNA-intercalating dye that is used to detect increased permeability of cell membranes, after antifungal treatment.31 It is know that compounds that cause propidium iodide influx may directly affect fungal membranes, leading to fungal cell death.32,33 Alterations in membrane integrity and function could be another mechanism associated with the synergistic interaction between promethazine and azole antifungals. An interesting fact noticed is that, at higher concentrations of promethazine (256 μg/mL), a decrease in intracellular fluorescence is observed, indicating a decrease in plasma membrane damage. However, as observed in the time-kill curve assay, higher concentrations of promethazine start kill C. tropicalis cells after 2 h incubation. This fact indicates that membrane damage is not the major effect leading to death when C. tropicalis cells are exposed to high concentrations of promethazine. Moreover, the mitochondrial function was altered when C. tropicalis cells were treated with promethazine, suggesting the interference of this compound on mitochondrial respiratory function. Decrease in Rh123 fluorescence, a cationic dye used to label mitochondria in living cells, is related to depolarization and de-energization of mitochondria, which leads to reduction in mitochondrial function.34,35 It is important to highlight that collapse of mitochondrial transmembrane potential can cause release of pro-apoptotic factors into the cytosol, leading to cell death.27 Moreover, specific drugs that target fungal mitochondria could act in synergy with triazoles since this organelle acts as an energy conduit that is needed for many cell processes, including ergosterol biosynthesis.36 In fact, a mitochondrial respiratory dysfunction leads to a decrease in NADPH and oxygen, which are respectively required for squalene dimerization and the conversion of squalene into 2,3 oxidosqualene, previous steps to that targeted by azoles.37 Thus, the synergism between azoles and promethazine may also be associated with the action of the latter on fungal mitochondria and cell structure, leading to the decrease in efflux activity and ergosterol biosynthesis, resulting in the reduction of the antifungal MICs against azole-resistant strains. Alterations in mitochondrial function and cell morphology were observed in both azole-resistant and azole-susceptible strains. However, synergism between azoles and promethazine was only observed against azole-resistant isolates. This difference could be associated with differences in cell metabolism, as azole-resistant strains commonly present increased metabolic activity, often associated with increased respiration and gene overexpression, including ERG11, CDR and MDR.6 Additionally, Denerdi et al.38 reported that the combination of tacrolimus, a calcineurin inhibitor, also known for its efflux pump inhibiting properties,39 and azoles showed high percentages of synergy against fluconazole-resistant C. glabrata but not against susceptible strains. Moreover, antagonistic interactions between tacrolimus and fluconazole were also observed against susceptible strains, similar to the results found in this study, where antagonistic interactions between promethazine and azoles (fluconazole and voriconazole) against the susceptible strain were observed. Therefore, these results corroborate those found in our study, since no synergistic interactions between promethazine and azole drugs were observed. In summary, the present study demonstrated the synergistic effect between promethazine and azoles against resistant C. tropicalis isolates, reversing the resistance phenotype to all tested azoles. Promethazine presented an antibiofilm activity at a concentration 4 times the MIC against planktonic cells. Promethazine showed antifungal activity, altering cell size/granularity, membrane integrity and mitochondrial function, which are potential mechanisms of promethazine-mediated cell damage against C. tropicalis cells. Supplementary material Supplementary data are available at MMYCOL online. Funding This work was supported by the National Council for Scientific and Technological Development (CNPq; Brazil; Process number: 443167/2014-1). Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Negri M, Silva S, Henriques M, Oliveira R. Insights into Candida tropicalis nosocomial infections and virulence factors. Eur J Clin Microbiol Infect Dis . 2012; 31: 1399– 1412. Google Scholar CrossRef Search ADS PubMed  2. Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J. Candida glabrata, Candida parapsilosis and Candida tropicalis: biology, epidemiology, pathogenicity and antifungal resistance. FEMS Microbiol Rev . 2012; 362: 288– 305. Google Scholar CrossRef Search ADS   3. Da Costa KRC, Ferreira JC, Komesu MC, Candido RC. 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Medical MycologyOxford University Press

Published: Oct 9, 2017

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