Fungicidal activity of human antimicrobial peptides and their synergistic interaction with common antifungals against multidrug-resistant Candida auris

Emergence of Candida auris, a multidrug-resistant yeast, demonstrates the urgent need for novel antifungal agents. Human antimicrobial peptides (AMPs) are naturally occurring molecules with wide spectrum antimicrobial activity, particularly against a variety of fungi. Therefore, this study examined the antifungal activity of seven different human AMPs against C. auris following the CLSI guidelines. The antifungal activity was further assessed using time kill curve and cell viability assays. For combination interaction, effectiveness of these peptides with three antifungals, fluconazole, amphotericin B, and caspofungin was done following standard protocols. To elucidate the antifungal mechanism, the effects of peptides on membrane permeability were investigated using propidium iodide staining method and confocal imaging. Antifungal susceptibility results showed that all the examined peptides possessed fungicidal effect against C. auris at different levels, with human β-defensin-3 being the most potent antifungal with MIC values ranging from 3.125 to 12.5 µg/ml. Time kill curves further confirmed the killing effect of all the tested peptides. Viability assay showed a significant decrease in the percentage of viable cells exposed to different inhibitory and fungicidal concentrations of each peptide (p < 0.01). Furthermore, peptides showed mostly synergistic interaction when combined with conventional antifungal drugs, with caspofungin showing 100% synergy when combined with different AMPs. As antifungal mechanism, peptides disrupted the membrane permeability at concentrations that correlated with the inhibition of growth. Overall, the findings of this study point towards the application of the tested peptides as a monotherapy or as a combination therapy with antifungal drugs to treat multidrug-resistant C. auris infections.


Introduction
Since Candida auris emergence in 2009 (Satoh et al. 2009), it has been identified as a causative agent of nosocomial infections in compromised individuals globally (Dahiya et al. 2020). This pathogen has been involved in colonization of multiple surfaces and equipment in healthcare facilities (Lockhart et al. 2017a) and is therefore responsible for several hospital outbreaks worldwide, with high mortality rates between 30 and 72% (Zhu et al. 2020).
Previous studies have reported that C. auris isolates are commonly resistant to azoles and polyenes (Osei Sekyere 2018). Echinocandins (e.g., caspofungin) exhibit a strong activity against C. auris isolates and currently used as a standard recommended treatment for these infections (Lyman et al. 2021). However, secondary resistance among previously susceptible strains has been observed ). These challenges have encouraged the urge to develop alternative antifungal compounds with a low potential for inducing fungal resistance. Therefore, the discovery of novel antifungals from natural sources, such as peptides produced by host defense system themselves, has been the focus of many studies (Mercer and O'Neil 2020).
Antimicrobial peptides (AMPs) are important constituents of the innate immune response produced in humans and most forms of life (Mahlapuu et al. 2016). Many AMPs typically 1 3 exhibit a wide range of activity protecting the host from different pathogenic fungi, bacteria, enveloped viruses, and protozoa (Hancock and Chapple 1999). Furthermore, they have immunomodulatory activity by attracting the phagocytic cells to the sites of infection (Choi et al. 2012). Therefore, natural and synthetic AMPs have been considered as potential alternatives to the traditional antimicrobial drugs (Kang et al. 2017).
In general, AMPs are a group of relatively small cationic polypeptides (Malmsten 2014), commonly classified into three large families including (i) α-helical, (ii) β-sheet, and (iii) extended peptides (Nguyen et al. 2011). The human defensins belong to β-sheet category and human salivary histatin 5 peptide, belong to extended peptide, and are of the major peptides presented in humans (Buda De Cesare et al. 2020). The human defensins are further divided into two subfamilies: human αand β-defensins (Schneider et al. 2005). Previously, different AMPs have been illustrated in several studies to have significant antifungal activities against different medically important fungal pathogens including Candida, Cryptococcus and Aspergillus species (Fernández de Ullivarri et al. 2020).
In the field of antifungal drug development, combination approach together with screening for novel antifungal molecules has been the focus of many researches in the hope of discovering better therapeutic option (Scorzoni et al. 2017). Combination antifungal therapy has become a common approach used to treat resistant fungal infections (Candoni et al. 2014). Combining an antifungal agent with another compound that has antifungal activity can overcome the development of drug resistance in pathogenic fungi by using lower dosage of both agents, with a concurrent reduction in associated side effects (Baddley and Pappas 2005). Several AMPs have shown promising synergetic activity when combined with commercial antifungals against Candida albicans (Krishnakumari et al. 2008) and other pathogenic non-Candida albicans (Lupetti et al. 2003;Singh et al. 2017;Wei and Bobek 2004).
Based upon the present knowledge of the antifungal properties of the major human AMPs, the present study evaluated the antifungal activity of seven peptides individually and combined with three common antifungal agents against clinical C. auris isolates. The results presented here have provided evidence of their promising role in the field of antifungal drug development.

Strains and growth conditions
Clinical C. auris isolates (n = 10) used in this study were previously identified and stored as glycerol stocks at − 70 °C in the Department of Clinical Microbiology and Infectious Diseases, University of the Witwatersrand. Each strain was initially grown on Sabouraud dextrose agar (SDA) plates and incubated at 37 °C for 24 h, and then pure colonies from these plates were suspended in 15 ml of Sabouraud dextrose broth (SDB) with continuous shaking at 200 rpm for 18 h. After that, cells were centrifuged, washed three times with phosphate buffer solution (PBS), adjusted to 1 × 10 6 CFU/ml and used for the subsequent assays.

Peptides and antifungal drugs
Seven peptides from the different classes of human AMPs, which are human neutrophil peptides 1-3 (HNP-1, HNP-2, and HNP-3), Histatin 5 (His 5), and human β-defensin 1-3 (hBd-1, hBD-2, and hBD-3), were used in this study. All peptides were purchased from Thermo Fisher Scientific, IL, USA. Mass spectrometric (MS) analyses of the peptides were performed using Applied Biosynthesis Voyager DE system, and purity of ≥ 95% was measured by high-performance liquid chromatography (HPLC) system (Biosynthesis, Inc.) as per the certificate of analysis provided by the supplier. All peptides were dissolved in distilled water to make stock solution of 5 mg/ml and kept in a dark environment at − 20 °C until use. The following antifungal drugs (fluconazole, FLC; amphotericin B, AmB and caspofungin, CAS) procured from Sigma-Aldrich (St. Louis, MO, USA) were also used in this study.

Antifungal susceptibility assay
Antifungal activity of the selected peptides and three antifungal drugs was determined using broth microdilution method as per Clinical and Laboratory Standard Institute (CLSI) guidelines reference document M27-A3 (CLSI 2008) with modification. AMPs (800 µg/ml) and antifungal drugs (200 µg/ml of FLC; 32 µg/ml of AmB; 16 µg/ml of CAS) at volume of 100 µl were added to the first well of the 96-well plates, which were then serially diluted in SDB medium. One hundred microliters of standardized Candida suspensions, which further diluted the concentrations by one-fold, were added to each well, and plates were incubated at 35 °C for 24 h. The final concentration ranged from 200 to 1.56 µg/ml for peptides, 500 to 4 µg/ml for FLC, 8 to 0.063 µg/m for AmB, and 4 to 0.031 µg/ml for CAS. A culture control containing SDB and culture, a media control containing only SDB, and negative control containing sterile water were also included. Minimum inhibitory concentration (MIC) was determined as the lowest concentration of the tested peptides/ antifungals at which no visible growth was observed. Following MIC determination, minimum fungicidal concentration (MFC) was determined as the lowest concentration which causes no visible growth on agar plates after sub-culturing for 24 h from wells.

Time-kill curves assay
To study killing curves of each peptide, the procedure described by Klepser et al. (1998) was followed. Two strains (sensitive and 1 3 MDR) were chosen based on the MICs for the antifungal drugs. Standardized Candida suspension was treated with the tested peptides at concentrations equal to their MIC/MFC levels for the strains to be tested. Cultures with and without the test compounds (controls) were incubated at 37 °C with agitation at 200 rpm for 48 h. One hundred-microliter samples were taken out at 0, 2, 4, 6, 8, 12, 24, and 48 h, and then immediately washed and diluted in PBS, and 20-µl samples were plated onto SDA plates. Plates were kept for 24 h at 37 °C for colony count determination. The colony forming unit per ml (CFU/ml) was calculated, and the average of three measurements was log 10 transformed and plotted against time for each peptide.

Candida cell counting and viability assay
To further quantify the killing effect of each peptide, total cell counting and viability were assayed by Muse™ cell analyzer (EMD Millipore, Germany), following the manufacturer's instructions. C. auris cells were treated with the tested peptides at their MIC/MFC concentrations and incubated at 37 °C for 4 h. Both negative (untreated cells) and positive (cells treated with 10 mM H 2 O 2 ) controls were also included. The treated cells were then washed and resuspended in PBS, and 20 µl of the cell suspension was added to 380 µl of count and viability reagent (EMD Millipore, USA), after 5-min incubation at room temperature, the sample was analyzed for total cell counting and viability.

Combination assay
The peptide-antifungal interaction was evaluated using the microdilution method (Ahmad et al. 2015). The assay was carried out in 96-well microplates by combining the 50 µl of tested peptides and 50 µl of antifungals (FLC, AmB and CAS) in a volume ratio of 1:1 against all the C. auris strains followed by serial dilution by transferring 100 µl as described in antifungal susceptibility assay. Following serial dilution, 100 µl of Candida suspension was added to each well, and the plates were incubated at 35 °C for 24 h. After incubation, the MIC for each antifungal agent in combination with all the peptides was defined as described above. The combination interaction was assessed by calculating the fractional inhibitory concentration index (FICI) using the following formula: The results interpretation was as follows: synergy (FICI ≤ 0.5), additive (FICI between 0.5 and 1.0), indifference (FICI between 1.0 and 4.0), and antagonistic (FICI > 4.0).

Assessment of plasma membrane permeability
The effect of the tested peptides on C. auris membrane integrity was assessed using propidium iodide (PI; Sigma-Aldrich) as described (Suchodolski et al. 2017), with modification. Standardized cell suspension was treated with the different peptides at MIC/MFC concentrations for 4 h; washed cells were then stained with 30 µM of PI. After 30 min incubation in the dark at room temperature, cells were washed and resuspended in 200 µl of BPS. Laser scanning confocal microscope-Zeiss LSM 780 with Airyscan detector was used for sample visualization and image acquisition. Both positive and negative controls were prepared as described above and included in this assay.

Statistical analysis
GraphPad Prism 5 was used for statistical analysis. The data were presented as the mean of three independent replicates (mean ± SD) and analyzed using one-way analysis of variance (ANOVA) with Dunnett's comparison test (p value < 0.05).

Susceptibility profile of C. auris isolate to peptides and antifungals
Susceptibility of 10 C. auris isolates to seven peptides was evaluated, and the MIC/MFC values are shown in Table 1. For comparison, three conventional antifungal agents were also included in this study, and the antifungal susceptibility profile is shown in Table 2. All peptides exhibited good antifungal potential against all C. auris isolates with variation in the activity levels. Based on the MIC results, the order of antifungal efficacy was hBD-3 > hBD-1 > hBD-2 > His 5 > HNP-1 > HNP-2 = HNP-3, with MIC value ranges of 3.125-12.5 µg/ml, 6.25-25 µg/ml, 12.5-50 µg/ml, 25-50 µg/ml, 25-100 µg/ml, and 50-100 µg/ml, respectively. In terms of MFC results, all peptides gave values of twofold higher than their corresponding MICs for all strains evaluated, suggesting that all peptides have fungicidal activity against C. auris. As for the standard antifungal agents, CAS was the most active antifungal (MIC range; 0.25-2 µg/ml). For AmB and FLC, the MIC values were found between 0.125 and 4 µg/ml and between 16 and 500 µg/ml, respectively. Based on the Centers for Disease Control and Prevention (CDC) tentative MIC Breakpoints (CDC 2019), out of the 10 tested C. auris isolates, 8, 5, and 1 were classified resistant to FLC (MIC ≥ 32 µg/ml), AmB (MIC ≥ 2 µg/ml), and CAS (MIC ≥ 2 µg/ml), respectively.
Based on this classification, 2 isolates (CAU-02 and CAU-09) were chosen to further study the fungicidal activity of the selected peptides. The CAU-02 strain showed sensitivity (the lowest MICs) to all antifungals, and CAU-09 strain showed resistance (the highest MICs) to all antifungal drugs tested.

Killing kinetics of peptides against C. auris
The antifungal activity of AMPs was further assessed to determine their killing kinetics at MIC and MFC concentrations (Fig. 1). The fungicidal effect of the examined peptides was defined as ≥ 3 log 10 reduction in CFU/ml (≥ 99.99 killing) from the starting inoculum. All peptides had reached the maximum fungicidal endpoint (100% killing) at their highest concentrations tested (MFC) at 12 h and 24 h of the test period in both strains. However, the onset of the fungicidal effect differed between the two strains. Among the tested peptides, hBD-1, -2, and -3 exhibited the most rapid activity by killing C. auris cells within 6 h of treatment at their MFC concentrations and after 12 h at their MIC concentrations for both strains. Histatin 5 also exhibited rapid fungicidal effect at 6 h and 24 h of incubation for MFC and MIC concentration respectively in the MDR strain. The sensitive strain was killed within 8 h at MFC concentration, while at the MIC level experienced no killing effect. The time kill curves for HNP1, -2, and -3 looked similar with almost no killing effect at MIC level in both strains, whereas at MFC values, the killing effect at 8 h and 12 h in the MDR and sensitive strains was observed. This correlates well with the susceptibility results and revealed the time-and dose-dependent killing activity of the selected peptides towards C. auris.   Each value represents the median of 3 independent experiments. Classification based on CDC guidelines (tentative MIC breakpoints); FLC (S < 32 µg/ml; R ≥ 32 µg/ml); AMP (S < 2 µg/ml; R ≥ 2 µg/ml); CAS (S < 2 µg/ml; R ≥ 2 µg/ml)

Peptides rapidly reduce the viability of C. auris cells
Peptides' effect on cell viability of susceptible and MDR C. auris isolates was determined by using Muse™ Count & Viability kit. This method provides the absolute cell count and viable cell percentage in the sample, whereas the reagent can differentiate between viable and dead cells by simultaneous staining both populations based on their selective permeability to the two DNA-binding dyes. The results showed that treatment with the test peptides resulted in decrease in the percentage of cell viability after 4 h exposure when compared to untreated control cells ( Fig. 2; Table 3). The untreated negative control showed 95.8% and 96.0% live cells, while the positive control showed 0.5% and 0.9% live cells in CAU-02 and CAU-09, respectively. For the susceptible strain (CAU-02), hBD-1, -2, and -3 demonstrated the best killing effects and reduced the cell viability by 71.7 to 99.4% after treatment with MIC and MFC concentrations, respectively. While HNP-1, -2, and -3 displayed the poorest killing effect and reduced the viability by 24.4 to 67.6% after treatment with the same concentrations. Likewise, hBD-1, -2, and -3 also demonstrated the best killing effects against the MDR strain (CAU-09) where the reduction in cell viability percentage was between 58.9 and 98.8% after treatment with MIC and MFC values respectively. However, HNP-1, -2, and -3 displayed better killing effect (50.5-78.0%) than in the susceptible strain after treatment with the same concentrations. These are in accordance with the previous results indicating that the hBD-3 is the most active peptide. Finally, all peptides significantly inhibit the survival of C. auris cells (p < 0.01) compared to untreated negative controls in both strains tested (Fig. 2). The viability profile of C. auris strains after treatment with different concentrations of peptides are represented in Figure S1.

Combination activity of peptides with antifungal drugs
Microbroth dilution assay allowed to assess the antifungal effect of the peptides-antifungals combinations against C. auris strains. From the FIC indices, it has been observed that the combinations of FLC and AmB with the peptides displayed varied interaction among the isolates tested  (Tables 4 and 5). Synergistic effects were observed in 90% of the isolates when AmB was combined with HNP-1, HNP-3, and His 5, while only 10% of the isolates showed additive interactions. The combinations of AmB with hBD-1, -2, and -3, and HNP-2 produced FIC indices of synergism in 100% of the isolates. The FLC-peptide combinations were synergistic, additive, and indifferent in 70 to 90%, 20 to 10%, and up to 10% of the isolates, respectively. The calculated FIC indices of the combination of CAS with all the peptides were well 0.5 or less, pointing towards synergistic activity in all 10 strains tested (Table 6). No antagonistic activity was detected for any combination. The MICs of antifungal drugs and peptides in combination decreased by up to 4-and eightfold when compared to their MICs alone. This indicates that the antifungal drugs in combination with the tested peptides inhibited the Candida growth at lower concentrations compared to the drugs alone.

Peptides disrupt the C. auris membrane permeability
The permeabilization effect of peptides on C. auris membrane was assessed using PI staining method. PI is a DNAstaining dye which cannot cross the intact membrane;  however, it is able to penetrate the cells with compromised membrane and stain the nucleic acid. As seen in Figs. 3 and 4, the negative control (untreated healthy cells) showed no fluorescence signal. However, the yeast cells showed a clear red fluorescence after 4 h of incubation with the different peptides as well as the positive control (cells exposed to H 2 O 2 ) in both strains. Figures 3 and 4 represent the impact of the most active peptides HNP-1, His 5, and hBD-3 on membrane permeabilization of the susceptible C. auris CAU-02 and MDR CAU-09 strains respectively. The impact of the other tested peptides HNP-2, HNP-3, hBD-1, and hBD-2 on the cell membranes of the susceptible C. auris CAU-02 and MDR CAU-09 strains are presented in Figures S2 and S3 respectively. The results suggested that the tested peptides were able to increase the permeability of C. auris membrane as shown by increasing PI fluorescence. Variation in the fluorescence intensity and the number of non-viable cells (PI-positive) was observed in a dose-dependent pattern in both sensitive and MDR strains, which correlates well with the other results in this study.

Discussion
The emergence of C. auris, the MDR species, emphasizes the need for new therapeutic strategies to control antifungal resistance (Lone and Ahmad 2019). Recently, new approaches influenced by the natural host immune defense against fungal pathogens represent potential alternatives to the classic antifungal therapy (Mercer and O'Neil 2020), and consequently, the development of novel antifungal formulations based on natural AMPs has increased (Buda De Cesare et al.   1 3 2020). Our findings revealed the ability of selected human AMPs to exert fungicidal potential towards C. auris. Among them, hBD-3 showed the best activity with the lowest MIC/ MFC of 6.25/12.5 µg/ml and the highest reduction in the percentage of cell viability by 68.4 and 99.4%. In addition, it showed 90 to 100% synergistic effect when combined with FLC, AmB, and CAS. These results are encouraging in view of the high demand for developing alternative and/or potentiating antifungal therapies.
The management of C. auris infections has become challenging due to growing resistance to common antifungals. Additional concern is that the available therapeutics are relatively few and quite cytotoxic (Dahiya et al. 2020). Therefore, designing innovative and safe alternative antifungal compounds is desperately required. Several natural and synthetic AMPs from different sources have shown to cause growth inhibition or killing of different bacteria and fungi (Ciociola et al. 2016;Huang et al. 2014;Lin et al. 2015;Maiti et al. 2014), including C. auris (Dal Mas et al. 2019;Kubiczek et al. 2020;Pathirana et al. 2018;Raber et al. 2021;van Eijk et al. 2020;Vicente et al. 2019). In this study, the peptides based on the template of natural human AMPs were examined for their antifungal potential towards C. auris. All the examined peptides (HNP-1, -2, and -3; hBD-1, -2, and -3; and His 5) exhibited fungicidal activity towards clinical C. auris isolates, including the MDR strains. The susceptibility of C. auris to Histatin 5 has already been reported (Pathirana et al. 2018), but there are no reports, as far as we know, on the activity of human defensins against this yeast.
The fungicidal activity of the tested peptides was further interrogated by performing time kill assay, cell viability study, and membrane permeability assay on susceptible C. auris CAU-02 and MDR CAU-09 strains. Analysis of time kill curves showed that peptides induced cell death which increased with the time and the concentration. The maximum fungicidal endpoints were almost identical in treatment with all peptides in both strains (susceptible and resistant); it seems that peptides at high concentration eradicated susceptible and resistant C. auris cells with similar efficiencies. To further verify the fungicidal activity of AMPs tested against C. auris isolates, quantitative evaluation of the cell viability after 4 h incubation with the different peptides at their active concentrations was done using Muse Count & Viability kits. We found that all tested peptides significantly kill C. auris clinical strains (p < 0.01) regardless of their drug resistance status, indicating potent fungicidal activity of these compounds.
Defensins represent a large family of AMPs contributing to the host defense response against invading pathogens. Human α-and β-defensins are predominantly produced by neutrophils and epithelial cells, respectively, and play a key role in protecting human skin and mucosal surfaces (Schneider et al. 2005). Many studies have been conducted to study the activity of human defensins against Candida albicans, since these peptides are characterized by broad-spectrum antimicrobial properties (Polesello et al. 2017). Our results showed that hBD-3 possesses high antifungal activity, which is comparable to the results described in previous studies showing that hBD-3 was more effective against C. albicans than hBD-1 and 2 (Krishnakumari et al. 2008;Vylkova et al. 2006). Since C. auris has shown good susceptibility to human β-defensins, future investigations of the potential fungicidal activity of these peptides against other different non-albicans Candida species will be beneficial to explore their potential use as antifungal lead compounds.
Our results also indicated that exposure of C. auris to histatin 5 had more potent antifungal activity in drug-resistant strain  1 3 activity of histatin 5 against C. auris presented here encourages further studies to explore its specific target sites in this yeast. Combination antifungal treatment is commonly used to treat invasive candidiasis that have not responded to monotherapy (Spitzer et al. 2017). For instance, combination of echinocandins with azoles or AmB has been proposed for invasive C. auris infection management (Fakhim et al. 2017;Jaggavarapu et al. 2020). The idea of combining two structurally and functionally different compounds appears to have strong potential in developing a successful therapeutic approach against multiple drug-resistant infections (Zimmermann et al. 2007). Apparent synergism between different AMPs and antifungal drugs against clinical isolates of C. albicans has been reported in vitro (Bondaryk et al. 2017)  and resulted in improved survival in animal models (Mac-Callum et al. 2013). Thus, we evaluated the combined action of AMPs with clinically approved antifungals against C. auris. We observed high levels of synergism in almost all the antifungal-peptide combinations. More pronounced synergy, however, was observed for CAS than for FLC and AmB. The mechanism of interaction, especially synergism, between AMPs and antifungals is unknown; further studies are needed to demonstrate the mechanism of these synergistic interactions. Additionally, the combinations remarkably decrease the MIC values for all the AMPs, and drugs in comparison with the active concentration of each AMP and drug alone were observed. These findings also highlight the importance of such approaches to decrease the toxicity and the likelihood of resistance developing against the classical antifungals. The results demand further in vivo investigation to prove these claims and to study the cytotoxicity effects of these AMPs on host cells. There are studies indicating that human peptides do not exhibit general cytotoxicity effect on the viability of human cells. These peptides have been shown to bind selectively with the microbial cells with no or minimal interaction with the host cells, and this selectivity is thought to be highly dependent on the cationic properties of these peptides, leading to high attraction towards negatively charged microbial membranes rather than the less negatively charged host cell membranes (Ebenhan et al. 2014;Helmerhorst et al. 1999). The antimicrobial mode of action for defensins is still under investigation. AMPs have been reported to target the microbial cell membrane lipid structure resulting in the disruption of membrane integrity, followed by formation of unspecific membrane pores leading to cytoplasmic leakage and cell death (Rautenbach et al. 2016). The peptides in this study also effected C. auris membrane permeability at a varying level, as evident by confocal microscopy analysis. This indicated that the observed fungicidal effect of the tested peptides is related to an altered cell membrane permeability of C. auris which leads to cell death. These results are in consistent with the recent studies where AMPs have been reported to cause physical disruption of fungal cell membranes and thereby causing cell leakages and cell death (Benfield and Henriques 2020;Struyfs et al. 2021). Similarly, human AMP belonging to the cathelicidines family has also been reported to have fungicidal and membrane-disruptive activity against C. auris (Rather et al. 2022;van Eijk et al. 2020). Interestingly, while the interaction of the AMPs with the microbial membrane is believed to be a crucial step in their antimicrobial activity, some AMPs are known to combine multiple mechanisms and interact with different microbial structures, which therefore potentiate their killing activity (Fernández de Ullivarri et al. 2020). This could explain the different activity levels of the tested peptides as evident from the susceptibility results.
This study demonstrated strong fungicidal activity of selected AMPs towards small number of C. auris clinical isolates from South Africa (African clade). It is also worth considering larger number of isolates including strains from the other different phylogenetic clusters (South American, East Asian, South Asian, and Iranian clades) for future studies, since high genomic variations between these clades have already been proved by the whole-genome sequence analysis (Chow et al. 2019;Lockhart et al. 2017b).

Conclusion
MDR C. auris has been associated with hospital-acquired invasive infections with high mortalities. Since the available treatment options are very limited and relatively toxic to host cells, this emphasizes the demand for development of new alternatives to treat these infections. The results described in this work have shown that the tested AMPs especially hBD-3 have fungicidal activity towards C. auris alone or combined with known antifungal agents. Therefore, this study suggests the potential of human β-defensins and other human AMPs to be used as novel antifungal agents or in combination therapies.