Ninety-nine clinical isolates of C. albicans and 105 of C. auris were included in this study. The isolates were recovered between 2016 and 2021 from 17 departments of Colombia, as part of the National Surveillance Program of the National Institute of Health, Bogota, Colombia. Of the isolates, 157 (77%) were recovered from invasive infections, mostly BSI, while the remaining 47 (23%) were colonizing isolates, recovered from urine, urinary catheters and body parts of patients including bilateral nares, ears, axillae, groin, oral cavity, and rectum. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) was used for the identification of all isolates. As reference strains per species, both clinical invasive isolates, C. albicans ATCC 10231 and a multidrug resistant isolate of C. auris H0059-13-2251 from Colombia, were included. This last isolate has a minimum inhibitory concentration (MIC) of ≥64 µg/ml for fluconazole and a MIC of 8 µg/ml for amphotericin B, as such is considered resistant to both antifungals (Escandon et al., 2022).

From the 106 C. auris isolates studied, 55 (51.9%) had data regarding the antifungal susceptibility against amphotericin B, given that this testing is carried out as part of the national surveillance of C. auris in Colombia led by the National Institute of Health (Escandon et al., 2022). From these 55 isolates, 18 (32.7%) were identified as being resistant to amphotericin B, since they had a MIC ≥2 µg/ml (CDC, 2022).

C14R, with sequence CSSGSLWRLIRRFLRR and molecular weight of 2006.37 g/mol (Bodenberger et al., 2018; Mildenberger et al., 2024), was synthetized commercially by Synpeptide Co., Ltd. (Shanghai, China), and shipped in a lyophilized form with a purity of 95%. This peptide consists of seven amino acid residues that are either hydrophobic, aromatic, or non-polar, and nine that are polar or positively charged, strictly separated on opposite sides of the predicted α-helix (Mildenberger et al., 2024). At arrival, the peptide was weighed and resuspended in 4 ml of sterile water, to obtain several tubes containing a stock solution with a concentration of 400 µg/ml. These stock solutions were frozen at -20°C and thawed immediately before experiments.

To determine the antifungal activity of C14R against C. albicans and C. auris clinical isolates, antifungal susceptibility testing, using broth microdilution, was carried out based on the Clinical and Laboratory Standards Institute (CLSI) guidelines, following the M27M44S protocol (CLSI, 2022). First, each isolate was cultured on Sabouraud dextrose agar (SDA) at 35°C for 24 h. From a single colony of each isolate, an inoculum, at a concentration of 1-5×10⁶ cells/ml, corresponding to the 0.5 McFarland standard, was prepared in 5 ml of sterile water. Subsequently, a 1:50 dilution was made from each cell suspension in sterile water. To achieve a final inoculum concentration of 1-5×10³ cells/ml, a final dilution of 1:20 was done in RPMI 1640 supplemented with 3-(N-morpholino) propanesulfonic acid (MOPS) (Sigma-Aldrich, St. Louis, MO, United States). In a 96-well round-bottom microplate, 100 µl of the final inoculum were transferred and mixed with 100 µl of each concentration of the C14R peptide, per duplicate. The range of peptide concentration, tested by 2-fold serial dilutions, was 0.390625 to 200 µg/ml diluted in RPMI-MOPs medium. The final volume in each well was 200 µl. Wells containing RPMI-MOPS alone and wells with inoculum but without the peptide were used as controls for sterility and growth, respectively. Plates were then incubated at 35°C for 24 h. The MIC of the peptide, determined visually, was defined as the lowest concentration of C14R that caused a significant decrease in growth (>50%) compared to the growth control. The antifungal susceptibility to fluconazole of all isolates was also determined following the M27M44S protocol from the CLSI (CLSI, 2022). Fluconazole (Pfizer Inc., New York, NY, USA) was used in concentrations ranging from 0.125 to 256 µg/ml. Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were used as reference strains, following the CLSI recommendations (CLSI, 2022). In all experiments, 20 µl of the final inoculum were plated on SDA to verify the purity of each cell suspension and colony counts, followed by incubation at 35°C for 24 h.

To determine antifungal synergism between C14R and fluconazole, the checkerboard method, which is based on the standardized CLSI guidelines, protocol M27M44S (CLSI, 2022), was used. This method is a broth microdilution technique performed in 96-well round-bottom microplates (Spitzer et al., 2017). Considering the arrangement of the plates (8 × 12 wells) and the final volume, concentration gradients were prepared for each of the compounds utilized to create a dual concentration gradient. For the peptide, a concentration gradient ranging from 1.5625 to 200 μg/ml was tested, by dispensing, per duplicate, 50 μl of each concentration from columns 3 to 12 (from lowest to highest concentration). For fluconazole, a range between 0.25 and 128 μg/ml was tested, by dispensing 50 μl of each concentration from rows A to H (from lowest to highest concentration). To prepare the cell suspensions, each isolate was cultured on SDA at 35°C for 24 h. Thereafter, an inoculum adjusted to a concentration of 1-5×10³ cells/ml was prepared in RPMI-MOPS, as described above. From this suspension, 100 μl were mixed with the dual concentrations of peptide-fluconazole, to obtain a final volume of 200 μl per well. Controls for fungal growth and for sterility of the culture medium were considered in each experiment. For this assay, two clinical isolates of C. albicans, two of C. auris and the reference strain of C. auris H0059-13-2251, were chosen, given that, based on the antifungal susceptibility results, they had high MIC values to the peptide and were resistant to fluconazole. The reference strain of C. albicans ATCC 10231 was not used for this experiment, as this strain is susceptible to fluconazole and presented a low MIC to C14R.

The capacity of C14R to inhibit the biofilm formation of C. albicans ATCC 10231 and C. auris H0059-13-2251 was assessed with two colorimetric methods. The first one, crystal violet (CV), measures biofilm biomass, while the second one, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) with menadione, measures metabolic activity (O'Toole et al., 2000; Jin et al., 2003). Initially, each isolate was growth on Sabouraud dextrose broth at 35°C in shaker at 100 rpm for 18 h. After centrifugation at 2500 rpm for 5 min, yeast cells were suspended in RPMI-MOPS medium and adjusted to a concentration of 1-5×10⁶ cells/ml. In a 96-well flat-bottom microtiter plate per colorimetric evaluation, CV or XTT-menadione, 100 µl of each the inoculum were transferred and mixed with 100 µl of each concentration of C14R (0.390625 to 200 µg/ml), per duplicate, and then incubated at 35°C for 24 h. After this period, the planktonic phase in the wells was aspirated, and the biofilms were washed twice with 200 μl of 1× phosphate-buffered saline (PBS). After these washing steps, the adherent cells within the biofilm were stained either with 200 µl of a 0.1% (w/v) CV solution for 15 min at room temperature or with 100 µl XTT-menadione for 2.5 h in the dark, adding the respective volume of the reagent to the wells. After staining, the solution in the plates with CV was aspirated, and the stained cells were washed twice with 200 μl of 1× PBS. Following this, the biofilms were left to dry for 24 h at room temperature. Once the plates were completely dried, the biofilms with CV were treated with 200 µl of 30% acetic acid for 15 min, after which the whole volume was transferred to a new microtiter plate. On the other hand, after staining with XTT-menadione, 80 µl of the solution were aspirated and transferred to a new microtiter plate. To finalize, the optical density (OD) of the solutions in the new plates, either with CV or XTT-menadione, was read at 560 nm and 490 nm, respectively, using an Asys Expert Plus ELISA reader (Biochrom, Ltd. Cambridge, United Kingdom). In each plate, controls include peptide-free and biofilm-free wells. Each experiment was performed three times in duplicate.

Planktonic growth rate of C. albicans ATCC 10231 and C. auris H0059-13-2251 after treatment with C14R was evaluated as reported previously (Pinilla et al., 2022). Briefly, each isolate was cultured on SDA at 35°C for 24 h, after which yeast cells were suspended in RPMI-MOPS medium to a final concentration of 1-5×10³ cells/ml, as described above. From the adjusted yeast suspension, 100 μl were added to plates containing 100 μl of C14R dissolved in RPMI-MOPS medium. Final peptide concentrations were adjusted to 3.125 µg/ml and 6.25 µg/ml for C. albicans and to 25 µg/ml and 50 µg/ml for C. auris, considering the antifungal susceptibility assay. These values correspond to one dilution less than the MIC values, and the MIC values of the peptide for each species, respectively. Cell suspensions of each species without the peptide were included as growth control. Subsequently, plates were incubated with orbital shaking at 100 rpm at 37°C for 48 h and OD readings at 600 nm were taken automatically every 60 minutes using the Bioscreen system (Thermo Labsystems Type FP-1100-C).

Cell morphology of C. albicans ATCC 10231 and C. auris H0059-13-2251 was examined following treatment with a concentration of C14R equivalent to one dilution less than the MIC values for each species, as determined by antifungal susceptibility testing. This protocol was performed as previously described (Torres et al., 2023). Briefly, each isolate was grown on SDA at 35°C for 24 h. Thereafter, a suspension of each isolate was prepared in distilled water and adjusted to a concentration of 1-5×10⁶ cells/ml. From this inoculum, 450 μl were added to a 1.5 ml Eppendorf tube, mixed with 50 μl of C14R to obtain the desired concentration (3.125 μg/ml for C. albicans and 25 μg/ml for C. auris), and incubated at 35°C for 24 h. Subsequently, cells were harvested and fixed in 2.5% glutaraldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 3 h at room temperature and then in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for an additional hour. Cells were washed with the same solution and fixed with 1% osmium tetroxide (Sigma-Aldrich, St. Louis, MO, USA), dehydrated using an acetone gradient, and embedded in resin. Finally, the cells were stained with uranyl acetate (Thermo Fisher Scientific Inc., Waltham, MA, USA), examined in a transmission electron microscope (TEM) (JEOL JEM-1400 Plus, Tokyo, TYO, Japan), and photographed with a Gatan camera (Pleasanton, CA, USA), at the Pathology Laboratory of the Santa Fe Foundation, in Bogotá. Untreated yeasts served as controls.

To experimentally demonstrate pore-formation capability of C14R in the C. albicans membrane, a permeabilization assay was performed as described before (Mildenberger et al., 2024). For this, four fluorescent dyes were used, namely fluorescein (FITC), propidium iodide, ATTO 488 alkyne and rhodamine phalloidin (Thermo Fisher Scientific Inc., Schwerte, Germany) at final concentration of 5 µl/ml in 1× PBS. These dyes have different molecular sizes ranging from 389 to 1250 Da (FITC < propidium iodide < ATTO 488 alkyne < rhodamine phalloidin). In brief, 10⁷ C. albicans ATCC 90028 cells in 200 µl RPMI broth supplemented with 12 µg/ml C14R were incubated at 37°C for 2 h. Ten minutes prior to the end of the 2 h incubation time, 100 µl of a 0.1% (w/v) solution of Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) was added and served as positive permeation control. After incubation, the cells were centrifuged at 11000 × g and the supernatant was discarded, washed with 1× PBS, and after the addition of 5 µl each fluorescent dye and 195 µl 1× PBS for 20 min, the cell suspension was centrifuged at 11000 × g for 2 min, respectively. Supernatants were discarded, and then the cells were fixated for 10 min using 4% (w/v) solution of paraformaldehyde (Carl Roth GmbH, Karlsruhe, Germany). Subsequently, the yeasts cells were washed three times with 1× PBS, resuspended in 200 µl 1× PBS, and transferred to a flat-bottomed polystyrene 96 wells microtiter plate. Fluorescence measurements were conducted at excitation wavelengths of 498 nm (FITC), 535 nm (propidium iodide), 500 nm (ATTO 488 alkyne), 540 nm (rhodamine phalloidin), and emissions of 517 nm (FITC), 617 nm (propidium iodide), 520 nm (ATTO 488 alkyne), and 565 nm (rhodamine phalloidin) with a Tecan SPARK microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Additionally, microscopic analyses were performed at 630× magnification using a Leica DMi8 fluorescence microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany). Cells remained untreated for negative controls.

For each species, the frequency of MICs, mode, and geometric mean MICs of C14R and of fluconazole were determined. Differences in MICs between invasive and colonizing isolates, per species, were established using a Mann-Whitney test. The Pearson correlation coefficient (ρ) was used to assess the association between the MIC values of the peptide and those of fluconazole. ECVs for C14R, per species, were estimated with ECOFFinder (RRID : SCR_018149), a freely available Microsoft Excel spreadsheet calculator, following the methodology described previously (Turnidge et al., 2006). ECOFFinder was also used to generate graphs of the MIC distributions per species. This method has been adopted by the CLSI as a standard method for ECV determination (CLSI, 2016; CLSI, 2022). Definition of fluconazole and amphotericin B resistance in C. auris was done according to the interpretation of the Centers for Disease Control and Prevention (CDC), while fluconazole resistance in C. albicans was defined following the clinical breakpoints established by the CLSI (CDC, 2022; CLSI, 2022).

In checkerboard tests, to assess synergism, numerous combinations of antimicrobial agents were evaluated, and conventionally, the fractional inhibitory concentration (FIC) index value from the most effective combination was calculated using the formula: FIC = (MIC of drug A in combination/MIC of drug A alone) + (MIC of drug B in combination/MIC of drug B alone). The combination of antifungal agents was considered synergistic when FIC ≤0.5 μg/ml, and antagonistic when FIC >4.0 μg/ml, and any value in between was interpreted as indifferent (Maurya et al., 2011).

Statistical analyses, growth curves and molecular dynamics simulations graphs were performed with GraphPad Prism 9 (RRID: SCR_002798) and Microsoft^® Excel^® (RRID : SCR_017294). When established, p-values <0.05 were considered statistically significant.

The geometric mean MIC of C14R against the 99 clinical isolates of C. albicans plus the reference strain of the species was 4.42 µg/ml, with a mode that was the fourth lowest concentration tested (3.125 µg/ml) with 24 isolates. In addition, the ECV 95% for the species was 12.5 µg/ml, which is only two dilutions more than the mode ( Figure 1A ). Altogether, this indicates that C14R has very good antifungal activity for clinical isolates of C. albicans.

Regarding C. auris, it was possible to determine that C14R has also a good antifungal activity for the species, although this was to some extent inferior to that observed against C. albicans isolates. Both the mode (6.25 µg/ml) and the ECV 95% (25 µg/ml) of C14R against the 105 clinical isolates of C. auris plus the reference strain of the species ( Figure 1B ) were one dilution higher than the mode and the ECV 95% of C. albicans. In addition, the geometric mean MIC (5.34 µg/ml) was slightly higher. However, statistically, the susceptibility of the isolates of each species to the peptide did not differ among species (p = 0.3752).

Notably, none of the isolates, neither of C. albicans nor of C. auris, was able to grow in the highest concentration of C14R tested (200 µg/ml). In addition, statistically, the susceptibility of the isolates of each species to the peptide did not differ according to the source of the isolates. The geometric mean MICs of invasive isolates compared to colonizing isolates was 4.77 µg/ml vs. 3.74 µg/ml for C. albicans (p = 0.4267) and 5.78 µg/ml vs. 3.53 µg/ml for C. auris (p = 0.2193), respectively.

While C14R displayed a potent antifungal activity against clinical isolates of both C. albicans and C. auris, the antifungal activity of fluconazole was much lower against C. auris. Not only the geometric mean MICs of this azole differ statistically among C. albicans and C. auris (0.58 µg/ml vs. 9.94 µg/ml, p <0.0001), but also the mode (0.125 µg/ml vs. 2 µg/ml, p <0.0001). In addition, 36 (34%) isolates of C. auris were identified as fluconazole resistant (MIC ≥32 µg/ml), while most isolates (85%) of C. albicans were identified as susceptible (MIC ≤2 µg/ml). In C. albicans, three isolates were susceptible dose-dependent (SSD) (MIC = 4 µg/ml) and 12 were resistant (MIC = 64 µg/ml) to fluconazole. No association was found between the susceptibility of C. albicans to C14R and fluconazole (ρ = 0.04598, p = 0.6497), nor in the susceptibility of C. auris to both agents (ρ = -0.1617, p = 0.0978). However, from the 36 fluconazole-resistant C. auris isolates, four (11.1%) had simultaneously a high MIC to C14R (≥50 µg/ml).

From the 18 C. auris isolates that were resistant to amphotericin B, nine (50%) had in addition concomitant resistance to fluconazole. However, from this multidrug-resistant isolates, eight had a low MIC (≤6.25 µg/ml) to C14R and only one presented a MIC of 50 µg/ml to the peptide, which indicates that the peptide has very good antifungal activity against these fluconazole-amphotericin B resistant C. auris isolates.

The MIC values for C14R and fluconazole against the reference strains of C. albicans ATCC 10231 were 6.25 µg/ml and 1 µg/ml, respectively, and for C. auris H0059-13-2251 were 50 µg/ml and 128 µg/ml, respectively.

Among the studied clinical isolates, two of C. albicans and three of C. auris presented high MIC values to C14R (≥50 µg/ml) and were concomitantly resistant to fluconazole (≥32 µg/ml) ( Table 1 ). However, when the isolates were grown with different combinations of these agents, a clear synergistic effect was noticed, as the MIC values of C14R decreased between two and five dilutions and the MIC values for fluconazole decreased between one and six dilutions, with most FIC indexes ≤0.5 μg/ml ( Table 1 ). Notably, the combinations of C14R and fluconazole tested with each isolate caused the complete killing of Candida cells in both species.

Treating C. albicans ATCC 10231 biofilms with 6.25 µg/ml of C14R, which equals the MIC, not only decreased the total biofilm biomass ( Figure 2A ) but it was also effective in killing about 85% of the yeasts embedded in the biofilms ( Figure 2B ). Similarly, in C. auris H0059-13-2251, a concentration of about 25 µg/ml of C14R, which is one dilution less than the MIC, decreased almost the total biofilm biomass ( Figure 2A ) and it was effective in killing about 90% of the yeasts embedded in the biofilms ( Figure 2B ). Notably, C. albicans is able to produce more biofilm that C. auris, as it has been reported before (Sherry et al., 2017).

C. albicans growth is completely inhibited when is treated with a concentration of C14R that equals the MIC (6.25 µg/ml), and it is much slower when is treated with one dilution less than the MIC (3.125 µg/ml), which suggests that the peptide might be fungicide against this species ( Figure 3A ). Without treatment, the cell suspension of C. albicans reaches double the OD in ~23 hours with a constant increase in turbidity, while with the lower dose of treatment (3.125 µg/ml), the growth of C. albicans does not double even after 48 hours.

Compared to the effect on C. albicans, C14R shows a less strong effect on C. auris, given that the growth of the last species is not completely inhibited by the peptide. During the first 24 hours, C. auris treated with 25 µg/ml or 50 µg/ml grows much slower than without treatment. However, while the growth of C. auris at a concentration of C14R that equals the MIC (50 µg/ml) remains lower to untreated cells, even after 48 hours, a smaller concentration of the peptide (25 µg/ml) does not affect the yeasts growth after 27 hours, but instead the growth of treated cells become higher than the growth control, which suggests that the peptide might be fungistatic against C. auris ( Figure 3B ).

After treatment with C14R, the ultrastructure of both C. albicans ATCC 10231 and C. auris H0059-13-2251 was clearly changed. In untreated cells, the cell wall was smooth, the structures of the cell wall and cell membrane were intact, and the cytoplasm was uniform and full ( Figures 4A, B ). In treated cells, disruption of the membrane, causing the cell to lose its regular spherical shape, was observed in both species ( Figures 4C, D ). In addition, particularly in C. albicans, the surface of the cell seemed rough and there were white spots in the cells, which make the cytoplasm looks uneven with a disordered cytoplasmic structure ( Figure 4C ).

A lipid composition mimicking the C. albicans membrane was simulated and served to assess anti-Candida C14R interactions. C14R has a net charge of 5+ provided by five positively charged residues, constituting the 31.25% of the sequences. This feature drives the first step into the mechanism of action of AMPs adsorption. Here, the electrostatic forces between the positively charged residues of C14R and the negative charge of the lipid’s phosphate heads play an essential key role. Once the peptide has adsorbed onto the membrane, it was located at one side of the surface membrane and it presented elevated structural stability during the whole molecular dynamics’ simulation with a high contribution of α-helix (~75%) and a lower contribution of disordered structure (~25%) ( Figure 5A ). Additionally, it showed a tight association with the membrane, being located at the water-membrane interface, and at some points of the simulation (20 ns to 40 ns and 75 ns to 100 ns) crossing the phosphates barrier ( Figure 5B ). During the 100 ns of simulation, the average surface area of the peptide interacting with the membrane was 1253.19Ų representing 61.13% of the total area (2049.92 Ų) of the peptide ( Figure 5C ), where it adopted a parallel orientation to the membrane surface locating its polar residues Arg and Ser to the solvent and more hydrophobic residues as Leu, Phe, and Ile, deeply buried in the membrane ( Figure 5D ). The orientation adopted by the peptide on the membrane of C. albicans ATCC 90028 facilitates the interaction of all residues by short-range Van der Walls interactions. Also, the amphipathic nature of C14R and the insertion of hydrophobic residues into the membrane allow the peptide-membrane complex stabilization by hydrophobic interactions mostly between Leu6, Trp7, Leu9, Ile10, Phe13, and Leu14, and the lipid’s tails. On the other hand, polar residues exposed to the solvent and in the interface can establish several hydrogen bond interactions, especially Cys1, Ser2, Ser5, Trp7, Arg8, Arg11, Arg15, and Arg16. In addition, most of the Arg residues and Cys1 are involved in cationic interaction through their positive charge and the negative charge of the lipid’s phosphate heads ( Figure 5E ). The fact that arginines are highly involved in both interactions, hydrogen bonds and cationic interactions, reinforces the idea that these positive charges are highly involved in driving the peptide toward the negative surface of C. albicans and its further stabilization through electrostatic forces.

In addition, the predicted pore-forming ability of C14R, assessed by a permeabilization assay, showed that the detergent Triton X-100 served as the ultimately pore-forming positive control agent ( Figure 6A ). While FITC and propidium iodide were able to enter the cells unhindered after treatment with C14R, ATTO 488 alkyne could only enter to a certain degree and rhodamine phalloidin was completely excluded from entering the cells ( Figure 6A ). This shows that the peptide not only forms pores, but also defines a certain size limit for molecules to enter the cytoplasm via this introduced gates. By fluorescence microscopy it was possible to verify that Triton X-100 permeabilized C. albicans cells perfectly, allowing all dyes to be internalized, while C14R-treated cells allowed the entrance only of FITC, propidium iodide and, to a lesser extent, ATTO 488 alkyne ( Figure 6B ).