The clinical isolates of fluconazole-susceptible and resistant Candida were obtained from Instituto Adolfo Lutz (São Paulo, Brazil): Candida albicans (n = 14), Candida parapsilosis (n = 9), Candida tropicalis (n = 10), Candida glabrata (n = 10), and Candida krusei (n = 10). Standard strains of C. albicans (SC5314, ATCC 10231 and ATCC 24433), C. parapsilosis (ATCC 22019), C. tropicalis (ATCC 200956 and ATCC 28707), C. glabrata (ATCC 2001) and C. krusei (ATCC 6258) were also included. All strains were maintained at -80°C. To perform the experiments, the fungi were subcultured in Sabouraud dextrose broth (Becton, Dickinson and Company; Sparks, NV, USA) at 37°C for 24 h at 150 rpm before each assay.

The biphosphinic cyclopalladated complex C7a was synthesized from N,N-dimethyl-1-phenethylamide (dmpa) and complexed to 1,2 ethanebis (diphenylphosphine, dppe) ligand, as previously described (Rodrigues et al., 2003). The compound was diluted to a final concentration of 10 mM in DMSO (cell culture tested, Sigma–Aldrich, St. Louis, MO, USA), and for in vitro and in vivo assays, diluted to the final concentration in culture media or PBS, respectively. For use in the vaginal candidiasis model, C7a (2.5 and 5% w/w) was incorporated into a vaginal cream (10% wax self-nonionic emulsifier, 2% mineral oil, 5% propylene glycol and 84% distilled water, pH 4.5) and applied topically (Rossi et al., 2012).

Three antifungal agents were used as references: amphotericin B (AMB; Sigma–Aldrich) diluted in DMSO to obtain the concentration of 1,600 μg/mL; miconazole (MCZ; Sigma–Aldrich) diluted in DMSO to obtain the concentration of 1,600 μg/mL; and fluconazole (FCZ; Pfizer, São Paulo, Brazil) diluted in sterile water to obtain the concentration of 2,560 μg/mL. The stock solutions were stored at -20°C. For in vitro and in vivo assays, antifungals were diluted to the final concentration in culture media or PBS, respectively. For use in the vaginal candidiasis model, MCZ (2% w/w) was incorporated into the vaginal cream described above.

The antifungal susceptibility of planktonic cells of Candida spp. clinical isolates and standard strains was assessed by broth microdilution assay as described in the protocols M27-A3 (Clinical, and Laboratory Standards Institute [CLSI], 2008) and M27-S4 (Clinical, and Laboratory Standards Institute [CLSI], 2012), according to the Clinical and Laboratory Standards Institute.

The IC₅₀ and IC₉₀ values were determined by spectrophotometric reading at 492 nm in a microtiter plate reader (Epoch, Biotek Instruments, USA). The minimum inhibitory concentration (MIC, Supplementary Table S1) was defined as the lowest concentration inhibiting 90% (IC₉₀, for polyene agents) and 50% (IC₅₀, for azole agents) of fungal growth (Clinical, and Laboratory Standards Institute [CLSI], 2008). For C7a compound, MIC values were defined as the IC₉₀. As fungal populations were higher than 10 strains, MIC₅₀ and MIC₉₀ could be determined and are defined as the concentrations that inhibit 50 and 90% of population, respectively (Table 1).

To evaluate the minimum fungicidal concentration (MFC), aliquots (10 μL) were taken from each well of the broth microdilution assay, plated in drug-free media (Sabouraud dextrose agar) and incubated at 37°C for 48 h. The MFC was defined as the lowest concentration that completely inhibited the formation of colonies. The cumulative percentage of isolates inhibited by C7a or AMB, the most effective compounds, for each dilution is presented in Table 2. For MIC and MFC determinations, each point was performed in triplicate. MIC and MFC results shown in Supplementary Table S1 represent the median value of 3 experiments executed.

Candida albicans ATCC 10231 yeasts (10³ CFU/mL) were treated with 0.5 μg/mL of C7a for 24 h at 37°C in RPMI 1640 media buffered with MOPS 0.16 M. After this period, C7a-treated and untreated cells were washed with PBS pH 7.2, fixed with 4% paraformaldehyde in PBS for 30 min, adhered to glass coverslips previously covered with poly-L-lysine and stained with Calcofluor-White 1 mg/mL (Sigma–Aldrich) for 5 min. The coverslips were washed and mounted in n-propyl gallate solution (1%) and observed under EVOS FL microscope (AMG, WA, EUA). For quantification, about 300 cells were counted in random fields in the untreated and C7a-treated groups. Result shows representative images of two independent experiments.

Candida albicans ATCC 10231 treated 24 h with 0.5 μg/mL C7a were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 2 h at room temperature. Post-fixation was carried out in 1% osmium tetroxide in cacodylate buffer containing 1.25% potassium ferrocyanide and 5 mM CaCl₂ for 2 h. Thereafter, the cells were dehydrated with increasing ethanol concentrations, 100% propylene and embedded in Spurr’s resin. Ultrathin sections were stained with uranyl acetate and lead citrate, and observed in a transmission electron microscope (model Jeol 100CX, JEOL, Japan).

The minimum concentration of C7a and AMB that inhibited the biofilm formation and preformed biofilm cell viability was verified as previously described (Pierce et al., 2008). To evaluate the effect of C7a and AMB in preventing the biofilm formation, 50 μL of each compound serially diluted in RPMI 1640 media (Sigma–Aldrich) buffered with 0.16 M MOPS pH 7.0, were added to plates containing 50 μL of C. albicans ATCC 10231 2 × 10⁶ CFU/mL in a 96-well plate. To evaluate the efficacy of the compounds against preformed biofilms, biofilms grown for 24 h were gently washed and 100 μL of serially diluted compounds was added. After the addition of C7a or AMB, the plates were sealed with parafilm and incubated at 37°C for 24 h. Plates were washed twice with PBS to remove non-adherent cells and the XTT viability test was performed to determine the metabolic activity of the biofilm. The positive control was the viability of untreated yeast cells and the negative control was the absorbance of the medium alone. BMIC was defined as the first concentration of C7a that reduced the metabolism of yeast cells in the biofilm compared to the positive control. Results show one representative experiment out of three independent experiments executed, samples in quadruplicates.

Isogenic BALB/c mice (6–8 weeks old females) were bred at the University of São Paulo, Brazil, in an animal facility under specific pathogen-free conditions. The procedures involving animals and their care were conducted according to the local ethics committee and international rules and all experiments were approved by the Institutional Animal Care and Use Committee of Institute of Biomedical Sciences (ICB), University of São Paulo (USP).

Disseminated candidiasis in immunocompetent animals was induced by intravenous inoculation (tail vein) of 3 × 10⁶ C. albicans ATCC 10231 yeast cells suspended in 100 μL of PBS on day 0. Mice were treated intraperitoneally (i.p.) with PBS (control), C7a (0.3 mg/kg) or FCZ (20 mg/kg) every day for 7 days, starting on day 1. Animals were sacrificed 8 days after infection and kidneys were collected for fungal burden determination. Results show one representative experiment out of three independent experiments executed.

For disseminated candidiasis in immunosuppressed animals, two doses of 100 mg/kg cyclophosphamide (Sigma–Aldrich) were administered i.p. 4 days and 1 day before infection with C. albicans, then again on day 3 post infection and every 4 days thereafter (Rossi et al., 2012). The animals were kept in cages lined with wood shavings and closed with an autoclaved filter, and served autoclaved food and water in order to maintain a sterile environment. Cages were exchanged twice a week in laminar flow hoods. The animals were considered anergic when the number of blood leukocytes was found to be fewer than 100 cells/mm³ (Andes et al., 2008). On day 0, animals were intravenously (i.v.) infected with 1 × 10⁴ C. albicans ATCC 10231 yeast cells suspended in 100 μL of PBS. Mice were treated i.p. with PBS (control), C7a (0.3 mg/kg) or FCZ (20 mg/kg), every day for 7 days, starting on day 1. The survival of the animals was evaluated for 30 days after infection. Results show one representative experiment out of three independent experiments executed.

For vaginal candidiasis, the pseudo-oestrus phase was induced by subcutaneous administration of 0.5 mg of 17 beta-estradiol valerate (Sigma–Aldrich) dissolved in sesame oil (Sigma–Aldrich) 3 days before vaginal infection (Hamad et al., 2004). Mice were infected intravaginally with 3 × 10⁶ C. albicans ATCC 10231 yeast cells suspended in 10 μL of PBS, on day 0. Topical treatment was performed with C7a and AMB incorporated in a vaginal cream, as described above, every day for 7 days, starting on day 1. The vaginas were collected on day 8 for fungal burden determination and histopathological analysis. Results show one representative experiment out of three independent experiments executed.

Mice were sacrificed 8 days after intravenous or vaginal infection and kidneys and vaginas, respectively, were removed and weighed. Tissues were individually homogenized by mechanical disruption in 1 mL of PBS and 100 μL of these suspensions were inoculated in plates containing brain-heart infusion (BHI) agar media. Colonies were counted visually after 24 h of incubation at 37°C.

The vaginal canal was excised 8 days after infection, fixed in 10% buffered formalin, and embedded in paraffin for sectioning. Sections were stained with Periodic acid–Schiff (PAS) and examined microscopically at 100× magnification (Optiphot-2, Nikon, Tokyo, Japan).

Statistical analyses were performed using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, USA). Statistical comparisons were made by analysis of variance (one-way ANOVA) followed by a Tukey-Kramer post-test. P-values of <0.05 indicated statistical significance. Survival curves were analyzed by the Log-rank (Mantel–Cox) test and p-values of ≤0.05 were used to indicate statistical significance. A 95% confidence interval was determined in all experiments.

Susceptibility of the clinical isolates and ATCC standard strains of C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, and C. krusei to C7a in vitro is compiled in Supplementary Table S1 and Tables 1, 2.

Supplementary Table S1 shows the MIC and MFC values for each isolate individually. Amongst clinical isolates and ATCC standard strains of all Candida species there are two multidrug-resistant strains (C. tropicalis ATCC 200956 and ATCC 28707) and one resistant isolate only to AMB (C. glabrata IAL-23); however, there are several isolates resistant to FCZ, including all C. krusei isolates (Supplementary Table S1).

Also of note is that FCZ- and MCZ-susceptible isolates (low MIC values) showed elevated MFC values for at least one of these drugs (>16 or >64), suggesting a fungistatic effect (Supplementary Table S1). Most of Candida strains were susceptible to low concentrations of C7a as well as AMB (MFC values are close to MIC values) suggesting a fungicidal action (Supplementary Table S1).

When all individual values of clinical isolates are grouped (All species, Table 1) and MIC values are evaluated, it is of notice that C7a and AMB showed a short MIC range, 0.25–4 μg/mL and 0.125–16 μg/mL, respectively. MCZ and FCZ showed a wider range of MIC values, 0.03–>16 μg/mL and 0.03->64 μg/mL, respectively, suggesting that the susceptibility/resistance response of clinical isolates to these azole compounds are highly variable (All species, Table 1). Individual Candida species showed similar results (Table 1). The fungicidal effect of the most active compounds, C7a and AMB, is shown as the cumulative percentage of MFC in Table 2. Considering grouped values of clinical isolates (all species, Table 2), C7a was more efficacious than AMB at low concentrations (0.25 and 0.5 μg/mL), however, at higher concentrations both compounds demonstrated similar effect killing most of isolates (≥80%). It is important to note that C7a was more efficacious against C. albicans than AMB: at concentrations of 0.25 and 1 μg/mL C7a killed 41.2 and 100% of isolates, respectively, whereas AMB at the concentration of 0.5 μg/mL eliminated only 11.7% of isolates and 100% of isolates were killed only in concentrations higher than 16 μg/mL (Table 2). However, AMB was more efficacious against isolates of C. parapsilosis (Table 2). For the other Candida species, the two compounds showed similar fungicidal efficacy (Table 2). When MFC values of compounds were compared, there was no significant difference between C7a and AMB (P = 0.8399, Supplementary Table S1). Significant differences were found when C7a was compared to FCZ or MCZ (P < 0.0001, Supplementary Table S1).

Treatment of C. albicans with 0.5 μg/mL C7a induced morphological modifications on yeast cells in vitro. Fluorescence microscopy with Calcofluor White of C7a-treated cells revealed that the compound was able to decrease (40%) the formation of hyphae/pseudohyphae compared to untreated cells (Figure 1E). This is supported by the observation of an intense staining of Calcofluor White in the yeast-mother cell in treated cultures (arrows, Figure 1D), while staining of untreated cells occurred at the end of hyphae corresponding to the cell apical growth region (arrows, Figure 1B). In addition, septa are formed in the untreated cells in contrast to the absence of septa in the C7a-treated cells (arrowheads in the Figures 2B,D, respectively). These results suggest that C7a affects the formation of hyphae/pseudohyphae by yeast cells in culture.

The morphological effects induced in C. albicans by the treatment with C7a were also evaluated using transmission electron microscopy which showed important alterations in some organelles and structures that may be potential targets for C7a. Untreated cells of Candida albicans ATCC 10231 presented a normal cell ultrastructure, such as compact and electron-dense cell wall, cytoplasmic membrane, homogeneous and electron-dense cytoplasm, nucleus and mitochondria (Figure 2A). Cells treated with 0.5 μg/mL of C7a for 24 h at 37°C exhibited a damaged cell wall (arrowheads in the Figure 2C). In addition, increased numbers of electron-lucent vacuoles (Figure 2D), DNA alterations as marginalization of genetic material in the nucleus (arrow in the Figure 2C) and apparent mitochondrial swelling were observed in C7a-treated cells (Figures 2B,C). Additional images are shown in Supplementary Figure S1.

The BMIC, minimum concentration of C7a that significantly reduced the metabolism of yeasts in a mature biofilm (Figure 3A) and reduced the biofilm formation (Figure 3B), was 1 μg/mL. Although effective, C7a was less effective than AMB which interfered with the mature biofilm and biofilm formation of C. albicans ATCC 10231 yeast cells in concentrations 8 and 4 times lower, respectively.

Previously immunosuppressed female BALB/c mice were injected in the tail vein with 1 × 10⁴ Candida albicans ATCC 10231 yeast cells, and were treated i.p. with 0.3 mg/kg of C7a or 20 mg/kg of FCZ every 24 h for 7 days. On day 8, the fungal load of treated and untreated animals was evaluated by quantifying the CFUs from recovered kidneys. The treatment with C7a or FCZ showed a significant reduction (P < 0.05) in the fungal burden when compared with untreated mice (Figure 4). Although a dose 66 folder high of FCZ was used (Rossi et al., 2012) the fungal burden reduction induced by both compounds was similar (Figure 4).

The effect of C7a treatment on survival of immunosuppressed animals infected systemically with Candida albicans was assessed. The group of infected animals treated with PBS reached 100% mortality 21 days post infection. Treatment of infected animals with 0.3 mg/kg of C7a produced a survival rate of 78% and treatment with 20 mg/kg of FCZ produced a survival rate of 100% within 30 days post infection (Figure 5).

Treatment of vaginal candidiasis with a topical cream containing 5% (but not 2.5%) of C7a or 2% of MCZ led to a significant (P < 0.05) decrease in vaginal CFU when compared to untreated mice (Figure 6). Histopathological analyses of vaginal canal tissue samples collected from BALB/c mice in which vaginal candidiasis was induced but not treated showed an established infection by Candida albicans, with several yeasts and the formation of pseudohyphae/hyphae (Figure 7A). A clear reduction in the vaginal fungal burden was observed in the group of animals treated with the topical cream containing MCZ 2% (Figure 7B). Mice submitted to treatment with a topical cream containing 5% of C7a also showed a robust decrease in the vaginal fungal burden when compared to the untreated group (Figure 7C).