Ceragenins are active against drug-resistant Candida auris clinical isolates in planktonic and biofilm forms

Ceragenins are active against drug-resistant Candida auris clinical isolates in planktonic and... Abstract Background Candida auris has emerged as a serious threat to human health. Of particular concern are the resistance profiles of many clinical isolates, with some being resistant to multiple classes of antifungals. Objectives Measure susceptibilities of C. auris isolates, in planktonic and biofilm forms, to ceragenins (CSAs). Determine the effectiveness of selected ceragenins in gel and cream formulations in eradicating fungal infections in tissue explants. Materials and methods A collection of 100 C. auris isolates available at CDC was screened for susceptibility to a lead ceragenin. A smaller collection was used to characterize antifungal activities of other ceragenins against organisms in planktonic and biofilm forms. Effects of ceragenins on fungal cells and biofilms were observed via microscopy. An ex vivo model of mucosal fungal infection was used to evaluate formulated forms of lead ceragenins. Results Lead ceragenins displayed activities comparable to those of known antifungal agents against C. auris isolates with MICs of 0.5–8 mg/L and minimum fungicidal concentrations (MFCs) of 2–64 mg/L. No cross-resistance with other antifungals was observed. Fungal cell morphology was altered in response to ceragenin treatment. Ceragenins exhibited activity against sessile organisms in biofilms. Gel and cream formulations including 2% CSA-44 or CSA-131 resulted in reductions of over 4 logs against established fungal infections in ex vivo mucosal tissues. Conclusions Ceragenins demonstrated activity against C. auris, suggesting that these compounds warrant further study to determine whether they can be used for topical applications to skin and mucosal tissues for treatment of infections with C. auris and other fungi. Introduction Candida auris has emerged as a global threat; since the initial report from Japan in 2009, it has spread and been isolated on five continents.1–6,C. auris can be a substantial cause of nosocomial infections in some settings and is associated with high levels of mortality.7,8 Of particular concern is the drug resistance found among many isolates. Resistance to azole antifungal agents with clinical isolates is common and resistance of some strains of C. auris to all three classes of commonly used antifungals (azoles, polyenes and echinocandins) has been observed.1,9 The emergence of this pathogen has led to efforts to better track and characterize infections1,6 and it provides a strong impetus for the development of novel antifungal agents active against drug-resistant organisms.10 Higher organisms have faced threats from fungal pathogens for hundreds of millions of years and evolutionary pressures have yielded antifungal innate immune functions effective against organisms in both planktonic and biofilm forms. Antimicrobial peptides (AMPs) constitute a key component of these innate immune defences and AMPs from a variety of sources have shown potent antifungal activities.11–14 The prevalence of AMPs, in organisms ranging from mammals to amphibians to insects to plants, suggests that AMPs retain antimicrobial activities over extended periods without generation of widespread resistance. Recognizing the antifungal activities of AMPs and their anticipated retention of potency with extensive use, substantial efforts have been made to develop antifungal AMPs for clinical use.13 Impediments to clinical use include the relatively high cost of large-scale preparation of peptide therapeutic agents compared with that of small molecules and the instability of linear peptides in the presence of ubiquitous proteases. In an effort to overcome the challenges associated with these peptide therapeutic agents, while retaining the same mechanisms of antimicrobial action, we have developed a class of non-peptide molecules, based on a common bile acid, that mimic the amphiphilic morphology common to AMPs.15,16 Molecules of this class, termed ceragenins (CSAs) (examples shown in Figure 1), can be prepared on a large scale and, because they are not peptide-based, ceragenins are not substrates for proteases. Lead ceragenins are broad-spectrum antimicrobials with activity against drug-resistant bacteria, including colistin-resistant Gram-negative bacteria.17,18 Ceragenins also mimic ‘secondary’ activities of AMPs associated with wound healing.19 Figure 1. View largeDownload slide Structures of selected ceragenins. Figure 1. View largeDownload slide Structures of selected ceragenins. Recently, antifungal activities of selected ceragenins were reported,20 with MICs of ceragenins comparable to or below those measured with amphotericin B, fluconazole and AMPs, omiganan and LL-37, for multiple strains of Candida albicans. Fungi in the genera of Paecilomyces, Cryptococcus, Aspergillus, Scedosporium, Rhizopus, Blastomyces and Apophysomyces also proved susceptible to the ceragenins tested.20 Atomic force microscopy images of C. albicans untreated and treated with ceragenin CSA-13 showed changes in surface morphology of cells treated with the ceragenin, which were interpreted as showing membrane activity.20 Studies of mechanisms of antifungal activities of AMPs have concluded that membrane perturbation plays a key role21–23 and increases in damage by reactive oxygen species (ROS) and attenuation of mitochondrial functions, leading to apoptosis, have been observed.24–27 In a recent study, magnetic nanoparticles adorned with either the AMP LL-37 or CSA-13 resulted in generation of ROS in Candida spp.28 Considering the need for novel antifungal agents in the face of the growing threat of C. auris infection, our objectives in the studies described herein were to determine the antifungal activity of selected ceragenins against isolates of C. auris collected by CDC and to compare these activities with those of representatives of the three major classes of antifungal agents. Activities measured included both MICs and minimum fungicidal concentrations (MFCs) on planktonic organisms. Because fungi may grow in sessile form in biofilms, we also measured the activities of selected ceragenins against established biofilm forms of C. auris. To better understand the antifungal activities of ceragenins, scanning electron microscopy (SEM) images of C. auris, with and without ceragenin treatment, were acquired. Although ceragenins are well-tolerated in a variety of routes of administration,29–32 topical application to skin or mucosal tissues to treat fungal infections represents an attractive use of the antimicrobials. To determine the potential for use in this venue, different formulations of ceragenins CSA-44 and CSA-131 were evaluated in an ex vivo porcine vaginal mucosal tissue model infected with C. albicans or C. auris and compared with nystatin or a commercial cream containing miconazole. Materials and methods Materials and fungal strains CDC (Atlanta, GA, USA) has catalogued over 100 clinical isolates of C. auris for use in determining susceptibility patterns and these were used in initial studies with ceragenin CSA-131. These isolates represent all four of the known clades of C. auris and originated from various countries. These isolates were characterized as susceptible or resistant to fluconazole and echinocandins based on the MICs published by CDC (https://www.cdc.gov/fungal/diseases/candidiasis/recommendations.html), although breakpoints specific for C. auris have not yet been established. Ten additional isolates of C. auris and one strain of C. albicans (ATCC 90028, Manassas, VA, USA) were used to further characterize susceptibility to other ceragenins. Ceragenins CSA-44, CSA-131, CSA-142 and CSA-144 were prepared as reported previously.33,34 Three antifungal drugs belonging to the three main classes of commonly used antifungal compounds were used in comparison: amphotericin B (Fisher Scientific, Pittsburgh, PA, USA), fluconazole and caspofungin (Sigma–Aldrich, St Louis, MO, USA). The culture media used were Sabouraud dextrose broth (SDB) or agar (SDA) and Roswell Park Memorial Institute medium 1640 (RPMI) (Sigma–Aldrich) buffered at pH 7.0 with 165 mM morpholinopropanesulfonic acid (MOPS) (Sigma–Aldrich). Published protocols for growth of Candida spp. have used either SDB or RPMI,35–38 and to observe any medium-dependent changes in susceptibility, MICs and MFCs were measured in both media. Determination of susceptibility profiles of planktonic fungi MICs of the C. auris strains were measured according to the broth microdilution protocol from the CLSI M27-A3 document.39 With the entire CDC collection, RPMI medium was used in the measurement; inocula of 0.5 × 103–2.5 × 103 cfu/mL were prepared and used to seed pre-prepared drug plates containing CSA-131. These plates were incubated at 35°C and read after 24 h. With the smaller collection (10 isolates), two media (SDB and RPMI) were used to evaluate the antifungal activity of all four classes of antifungal compounds used in this study. MFCs were determined by plating 10 μL from each well of the MIC plate (after 24 h incubation) on SDA plates and measuring the lowest concentration of the antibiotic which eliminated 99.9% (3 logs reduction) of the colonies formed on the plates after 48 h of incubation at 35°C.40 Determination of susceptibility profiles of fungal biofilms To quantify the biofilm formed by clinical isolates of C. auris and determine the sessile susceptibility profile of ceragenins, metabolic activity within biofilms was imaged using XTT as described by Moss et al.41 In this assay, metabolic activity of cells in the biofilm is measured based on their reduction of XTT. Initially, biofilms were formed in sterilized polystyrene flat-bottomed 96-well microtitre plates (Sarstedt, USA) incubated at 35°C for 48 h and washed three times with PBS (Sigma–Aldrich) to remove planktonic cells. Ceragenins were then added to the wells in a concentration range of 1–128 mg/L and incubated for 24 h. A 10 mM menadione (Sigma–Aldrich) solution was prepared in 100% acetone and added to an XTT solution (0.5 mg/mL) to achieve a final concentration of 1 μM menadione. Aliquots of the XTT/menadione solution (100 μL) were added to each well containing biofilm and negative control wells. Plates were wrapped in aluminium foil and incubated at 37°C for 2 h. Aliquots of the supernatant (70 μL) were removed from each well and the OD was measured at 490 nm using a microtitre plate reader. Corresponding OD values for each strain were subtracted from the negative control values to calculate sessile MICs (SMICs). The SMIC50 and SMIC80 represent the concentration of the drug where the absorbance of the biofilm decreased by 50% or by 80%, respectively, compared with the biofilm formed by the same strain in the absence of the drug. Results are from three independent experiments. SEM of C. auris To observe the effect of ceragenins on cell membranes, C. auris CDC390 was cultured to mid-log phase and washed three times with PBS. Fungi were resuspended in PBS (OD600 = 0.2). CSA-131 (25 or 50 mg/L) was added and the mixtures were incubated at 37°C for 1 h. A control was prepared by incubating the fungal suspension without adding CSA-131. After collection via centrifugation, cells were washed with PBS three times. Glutaraldehyde (2.5%) in PBS was added to fix the cells at 4°C overnight. Resulting material was washed five times with PBS at 5000 rpm for 10 min using a microhaematocrit centrifuge (Hettich Mikro 20, Hettich, Tuttlingen, Germany) to remove the glutaraldehyde. Osmium tetroxide (0.5 mL) was used as a second fixative reagent and samples were stored at room temperature for 2–3 h. Cells were washed with PBS five times at 14 000 rpm for 8 min. A graded ethanol series (10%–100%) was used to dehydrate the cells. Samples were collected by centrifugation and the supernatant was discarded after each cycle. Finally, the samples were exposed to air in a desiccator at room temperature for 24 h. Dried fungal specimens were sputter-coated with 5–10 nm of a gold/palladium alloy and visualized under a scanning electron microscope (FEI Helios NanoLab 600 SEM/FIB, Hillsboro, OR, USA). Confocal laser scanning microscopy To observe the effects of treatment of fungal biofilms with CSA-131, aliquots (300 μL) of C. albicans and C. auris CDC383 (106 cfu/mL) were placed into separate single wells in a Lab-Tek® Chamber Slide™ (Nunc, Inc., Naperville, IL, USA) and incubated for 48 h. The biofilms were treated with CSA-131 (50 μL of a 50 mg/L solution) and incubated for 24 h at 35°C. After incubation, the solution was carefully removed and the wells were washed three times with PBS. Biofilms were stained with BacLight Live/Dead Viability Kit (L13152, Molecular Probes, Inc) according to the manufacturer’s instructions and observed at ×60 magnification using a confocal laser scanning microscope (Olympus FLUOVIEW FV1000). Assays in ex vivo tissue Ex vivo experiments were performed using porcine vaginal mucosal explants as previously described.42 Normal healthy porcine vaginal tissue was excised from animals at slaughter (Theurer’s Quality Meats, Lewiston, UT, USA) and transported to the laboratory on ice. Tissue was trimmed and collected in RPMI-1640 medium supplemented with penicillin (50 IU/mL, MP Biomedicals, Solon, OH, USA) and streptomycin (50 IU/mL, MP Biomedicals). Antibiotics were included to decolonize normal flora, which may interfere with biofilm formation. Explants of uniform size were obtained using a 5 mm biopsy punch. Excess muscle was trimmed away with a scalpel. After antibiotic washout (three changes of RPMI-1640 medium followed by 30 min incubation at 37°C), explants were transferred mucosal side up onto a polyethylene terephthalate 0.4 μm cell culture insert (BD Falcon, Franklin Lakes, NJ, USA) and inoculated with 2 μL (105 cfu/explant) of C. auris (CDC390) or C. albicans (ATCC 90028) prepared from overnight cultures in Todd Hewitt broth. Following 2 and 24 h incubation at 37°C, explants (n = 3) were treated with CSA-44L or CSA-131L (100 μL, 0.5% active) or CSA-44H and CSA-131H (100 μL, 2.0% active) in a hydroxyethylcellulose (HEC) gel formulation and subsequently incubated for 24 h at 37°C. For studies of cream formulations, aliquots of the test articles (100 μL) were spread evenly over explants. After treatment, tissues were suspended in a neutralizing broth [250 μL, Dey/Engley (HiMedia Laboratories, West Chester, PA, USA)], vortex-mixed (highest setting for 4 min) and then serially diluted in PBS and plated. Samples were spread on tryptic soy agar containing 5% sheep blood (BD Falcon) using a spiral plater (Microbiology International, Frederick, MD, USA) then incubated for 24 h at 37°C. The cream vehicle was assayed for antifungal activity by placing aliquots (100 μL) in a 96-well plate and adding fungal inocula (C. albicans ATCC 90028, 100 μL of 103, 104 or 105 cfu/mL cultures). Resulting samples were incubated (37°C) for 24 h. Aliquots from each sample were plated, incubated for 48 h and counted. Results were compared with controls that were not treated with the cream vehicle. Statistical analysis Analyses of variance (ANOVA) were performed by Dunnett’s multiple comparison post-test using the GraphPad PRISM software (GraphPad Software, Inc., La Jolla, CA, USA). Results and discussion Susceptibility of planktonic fungi to ceragenins The susceptibilities of 100 clinical isolates to a lead ceragenin, CSA-131, were determined and the distribution of CSA-131 MIC values ranged from 0.5 to 1 mg/L (Table 1). The overall mode was 1 mg/L and both the MIC50 and the MIC90 were 1 mg/L. CSA-131 showed activity against all four clades of C. auris with no variation in activity between the clades. There was no loss in activity for those isolates that were previously determined to be resistant to fluconazole and/or an echinocandin. Table 1. MICs of CSA-131 for fluconazole-resistant, fluconazole-susceptible and echinocandin-resistant C. auris isolates     MIC (mg/L)   C. auris isolate  n  range  mode  MIC50  MIC90  Fluconazole-susceptible  30  0.5–1.0  NA  0.5  1.0  Fluconazole-resistant  70  0.5–1.0  1.0  1.0  1.0  Echinocandin-resistant  7  0.5–1.0  1.0  1.0  1.0      MIC (mg/L)   C. auris isolate  n  range  mode  MIC50  MIC90  Fluconazole-susceptible  30  0.5–1.0  NA  0.5  1.0  Fluconazole-resistant  70  0.5–1.0  1.0  1.0  1.0  Echinocandin-resistant  7  0.5–1.0  1.0  1.0  1.0  NA, not applicable (bimodal with equal numbers of MICs of 0.5 and 1.0 mg/L). Table 1. MICs of CSA-131 for fluconazole-resistant, fluconazole-susceptible and echinocandin-resistant C. auris isolates     MIC (mg/L)   C. auris isolate  n  range  mode  MIC50  MIC90  Fluconazole-susceptible  30  0.5–1.0  NA  0.5  1.0  Fluconazole-resistant  70  0.5–1.0  1.0  1.0  1.0  Echinocandin-resistant  7  0.5–1.0  1.0  1.0  1.0      MIC (mg/L)   C. auris isolate  n  range  mode  MIC50  MIC90  Fluconazole-susceptible  30  0.5–1.0  NA  0.5  1.0  Fluconazole-resistant  70  0.5–1.0  1.0  1.0  1.0  Echinocandin-resistant  7  0.5–1.0  1.0  1.0  1.0  NA, not applicable (bimodal with equal numbers of MICs of 0.5 and 1.0 mg/L). To verify that other lead ceragenins also demonstrated activity against C. auris, studies were performed with a smaller collection of isolates and activities were compared with those of a strain of C. albicans. Both MICs and MFCs were measured in SDB or RPMI of CSA-44, CSA-131, CSA-142, CSA-144 and representatives of the three major classes of antifungal agents. As observed with the larger collection of C. auris isolates, MICs of CSA-131 were either 0.5 or 1.0 mg/L and MICs of CSA-44 and CSA-144 were similar (Table 2). MICs of CSA-142 were relatively higher. The strains tested were associated with relatively high MICs of fluconazole and one of the strains (CDC383) showed resistance to caspofungin. Compared with caspofungin, amphotericin B and fluconazole, the ceragenins gave lower MFCs, suggesting greater fungicidal activity compared with these other antifungal agents. Among the representative antifungal agents, caspofungin is the most closely related to ceragenins; it is a cationic molecule appended with a lipid chain. Similarly, ceragenins are cationic and a lipid chain is required for activity. Nevertheless, echinocandin-resistant C. auris strains (CDC383 for example) were fully susceptible to ceragenins (Table 1). In general, the medium in which MICs and MFCs were determined did not play a substantial role; MICs of the majority of the antifungal agents were identical in both media, as were MFCs. Table 2. Comparison of the susceptibility of clinical isolates of C. auris to selected ceragenins and three major classes of antifungal agents in SDB and RPMI   MIC (MFC) mg/L   Strains  CSA-44  CSA-131  CSA-142  CSA-144  CPF  AMB  FLC  SDB  SDB  SDB  SDB  SDB  SDB  SDB  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  Candida auris   CDC381  0.5 (2.0)a  0.5 (8.0)a  4.0 (32)a  0.5 (2.0)  2.0 (64)a  1.0 (48)  16 (>100)  [1.0 (8.0)]  [1.0 (64)]  [32 (>100)]   CDC382  0.5 (4.0)a  0.5 (8.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC383  0.5 (8.0)  1.0 (10)  2.0 (64)a  1.0 (8)a  32 (64)  1.0 (32)  64(>100)  [0.5 (16)]  [0.5 (8.0)]  [16 (48)]  [1.0 (64)]  [>100 (>100)]   CDC384  0.5 (4.0)a  0.5 (4.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC385  0.5 (16)a  0.5 (4.0)a  8.0 (32)a  1.0 (8.0)a  NM  NM  NM   CDC386  0.5 (8.0)a  1.0 (8.0)  4.0 (32)a  0.5 (8.0)  2.0 (64)a  2.0 (48)  64(>100)  [0.5 (8.0)]  [1.0 (16)]  [1.0 (64)]  [>100(>100)]   CDC387  0.5 (8.0)a  0.5 (8.0)a  4.0 (32)a  0.5 (8)a  NM  NM  NM   CDC388  1.0 (8.0)a  0.5 (4.0)a  4.0 (24)a  2.0 (8.0)a  NM  NM  NM   CDC389  0.5 (8.0)a  0.5 (8.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC390  0.5 (8.0)  0.5 (4.0)  4.0 (16)a  1.0 (8.0)a  2.0 (100)a  4.0(64)a  64 (>100)  [1.0 (32)]  [0.5 (8.0)]  [>100 (>100)]  Candida albicans   ATCC 90028  0.5 (8.0)  0.5 (4.0)  2.0 (8.0)a  2.0 (8.0)a  2.0 (32)  2.0 (100)  24 (>100)  [0.5 (16)]  [0.5 (8.0)]  [1.0 (8.0)]  [2.0 (64)]  [32 (>100)]    MIC (MFC) mg/L   Strains  CSA-44  CSA-131  CSA-142  CSA-144  CPF  AMB  FLC  SDB  SDB  SDB  SDB  SDB  SDB  SDB  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  Candida auris   CDC381  0.5 (2.0)a  0.5 (8.0)a  4.0 (32)a  0.5 (2.0)  2.0 (64)a  1.0 (48)  16 (>100)  [1.0 (8.0)]  [1.0 (64)]  [32 (>100)]   CDC382  0.5 (4.0)a  0.5 (8.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC383  0.5 (8.0)  1.0 (10)  2.0 (64)a  1.0 (8)a  32 (64)  1.0 (32)  64(>100)  [0.5 (16)]  [0.5 (8.0)]  [16 (48)]  [1.0 (64)]  [>100 (>100)]   CDC384  0.5 (4.0)a  0.5 (4.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC385  0.5 (16)a  0.5 (4.0)a  8.0 (32)a  1.0 (8.0)a  NM  NM  NM   CDC386  0.5 (8.0)a  1.0 (8.0)  4.0 (32)a  0.5 (8.0)  2.0 (64)a  2.0 (48)  64(>100)  [0.5 (8.0)]  [1.0 (16)]  [1.0 (64)]  [>100(>100)]   CDC387  0.5 (8.0)a  0.5 (8.0)a  4.0 (32)a  0.5 (8)a  NM  NM  NM   CDC388  1.0 (8.0)a  0.5 (4.0)a  4.0 (24)a  2.0 (8.0)a  NM  NM  NM   CDC389  0.5 (8.0)a  0.5 (8.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC390  0.5 (8.0)  0.5 (4.0)  4.0 (16)a  1.0 (8.0)a  2.0 (100)a  4.0(64)a  64 (>100)  [1.0 (32)]  [0.5 (8.0)]  [>100 (>100)]  Candida albicans   ATCC 90028  0.5 (8.0)  0.5 (4.0)  2.0 (8.0)a  2.0 (8.0)a  2.0 (32)  2.0 (100)  24 (>100)  [0.5 (16)]  [0.5 (8.0)]  [1.0 (8.0)]  [2.0 (64)]  [32 (>100)]  CAS, caspofungin; AMB, amphotericin B; FLC, fluconazole; NM, not measured. a Same result in both media. Table 2. Comparison of the susceptibility of clinical isolates of C. auris to selected ceragenins and three major classes of antifungal agents in SDB and RPMI   MIC (MFC) mg/L   Strains  CSA-44  CSA-131  CSA-142  CSA-144  CPF  AMB  FLC  SDB  SDB  SDB  SDB  SDB  SDB  SDB  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  Candida auris   CDC381  0.5 (2.0)a  0.5 (8.0)a  4.0 (32)a  0.5 (2.0)  2.0 (64)a  1.0 (48)  16 (>100)  [1.0 (8.0)]  [1.0 (64)]  [32 (>100)]   CDC382  0.5 (4.0)a  0.5 (8.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC383  0.5 (8.0)  1.0 (10)  2.0 (64)a  1.0 (8)a  32 (64)  1.0 (32)  64(>100)  [0.5 (16)]  [0.5 (8.0)]  [16 (48)]  [1.0 (64)]  [>100 (>100)]   CDC384  0.5 (4.0)a  0.5 (4.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC385  0.5 (16)a  0.5 (4.0)a  8.0 (32)a  1.0 (8.0)a  NM  NM  NM   CDC386  0.5 (8.0)a  1.0 (8.0)  4.0 (32)a  0.5 (8.0)  2.0 (64)a  2.0 (48)  64(>100)  [0.5 (8.0)]  [1.0 (16)]  [1.0 (64)]  [>100(>100)]   CDC387  0.5 (8.0)a  0.5 (8.0)a  4.0 (32)a  0.5 (8)a  NM  NM  NM   CDC388  1.0 (8.0)a  0.5 (4.0)a  4.0 (24)a  2.0 (8.0)a  NM  NM  NM   CDC389  0.5 (8.0)a  0.5 (8.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC390  0.5 (8.0)  0.5 (4.0)  4.0 (16)a  1.0 (8.0)a  2.0 (100)a  4.0(64)a  64 (>100)  [1.0 (32)]  [0.5 (8.0)]  [>100 (>100)]  Candida albicans   ATCC 90028  0.5 (8.0)  0.5 (4.0)  2.0 (8.0)a  2.0 (8.0)a  2.0 (32)  2.0 (100)  24 (>100)  [0.5 (16)]  [0.5 (8.0)]  [1.0 (8.0)]  [2.0 (64)]  [32 (>100)]    MIC (MFC) mg/L   Strains  CSA-44  CSA-131  CSA-142  CSA-144  CPF  AMB  FLC  SDB  SDB  SDB  SDB  SDB  SDB  SDB  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  Candida auris   CDC381  0.5 (2.0)a  0.5 (8.0)a  4.0 (32)a  0.5 (2.0)  2.0 (64)a  1.0 (48)  16 (>100)  [1.0 (8.0)]  [1.0 (64)]  [32 (>100)]   CDC382  0.5 (4.0)a  0.5 (8.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC383  0.5 (8.0)  1.0 (10)  2.0 (64)a  1.0 (8)a  32 (64)  1.0 (32)  64(>100)  [0.5 (16)]  [0.5 (8.0)]  [16 (48)]  [1.0 (64)]  [>100 (>100)]   CDC384  0.5 (4.0)a  0.5 (4.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC385  0.5 (16)a  0.5 (4.0)a  8.0 (32)a  1.0 (8.0)a  NM  NM  NM   CDC386  0.5 (8.0)a  1.0 (8.0)  4.0 (32)a  0.5 (8.0)  2.0 (64)a  2.0 (48)  64(>100)  [0.5 (8.0)]  [1.0 (16)]  [1.0 (64)]  [>100(>100)]   CDC387  0.5 (8.0)a  0.5 (8.0)a  4.0 (32)a  0.5 (8)a  NM  NM  NM   CDC388  1.0 (8.0)a  0.5 (4.0)a  4.0 (24)a  2.0 (8.0)a  NM  NM  NM   CDC389  0.5 (8.0)a  0.5 (8.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC390  0.5 (8.0)  0.5 (4.0)  4.0 (16)a  1.0 (8.0)a  2.0 (100)a  4.0(64)a  64 (>100)  [1.0 (32)]  [0.5 (8.0)]  [>100 (>100)]  Candida albicans   ATCC 90028  0.5 (8.0)  0.5 (4.0)  2.0 (8.0)a  2.0 (8.0)a  2.0 (32)  2.0 (100)  24 (>100)  [0.5 (16)]  [0.5 (8.0)]  [1.0 (8.0)]  [2.0 (64)]  [32 (>100)]  CAS, caspofungin; AMB, amphotericin B; FLC, fluconazole; NM, not measured. a Same result in both media. SEM of ceragenin-treated fungi As described above, membrane disruption has been identified as a major mechanism of antifungal activity of AMPs,21–23 among other possible mechanisms. Changes in cell morphology upon treatment with AMPs have been attributed to membrane interactions; these include loss of smooth cell surfaces with marked invagination at high AMP concentrations. Similar morphological changes in C. albicans were observed with the human AMP, LL-37, and the ceragenin, CSA-13.20 The impacts of CSA-131 on the morphology of C. auris are shown in Figure 2. As with C. albicans treated with LL-37 or CSA-13, CSA-131 alters cell shape; at 50 mg/L, CSA-131 causes cells to buckle in upon themselves and some cells appear to have merged. These changes in cell morphology were observed at concentrations well above MFCs and antifungal activity may be due to more subtle changes in cell permeability at lower concentrations of the ceragenin. At MFCs, little or no morphological changes in fungal cells were apparent. Figure 2. View largeDownload slide Scanning electron photomicrograph of untreated C. auris CDC390 (a and b) and treated with 25 mg/L CSA-131 (c and d) and 50 mg/L CSA-131 (e and f). Figure 2. View largeDownload slide Scanning electron photomicrograph of untreated C. auris CDC390 (a and b) and treated with 25 mg/L CSA-131 (c and d) and 50 mg/L CSA-131 (e and f). Susceptibility of fungal biofilms to ceragenins The abilities of bacteria and fungi to form biofilms, in which sessile organisms reside, contribute to drug resistance and protection of persister organisms that provide sources for chronic infections. The abilities of C. albicans to form biofilms are well-established;43–46 however, differing results for biofilm-forming propensities of C. auris have been reported.47,48 Using a measure of metabolic activity, we compared biofilm-forming properties of a C. albicans reference strain with those of four isolates of C. auris (CDC381, CDC383, CDC386 and CDC390). Biofilms were grown under identical conditions and, as reported,47C. auris strains were less efficient than the C. albicans strain at biofilm formation. Using the XTT-based assay, metabolic activity of the C. auris biofilms was measured as approximately 50% of that of C. albicans (data not shown). Having established the biofilm-forming characteristics of C. auris isolates, we used metabolic activity of biofilms to determine the activities of representative ceragenins and antifungals against sessile organisms making up these biofilms. Concentrations necessary to reduce biofilms by 50% and 80% are shown in Table 3. The ceragenins, amphotericin B and caspofungin demonstrated strong activity against the biofilms, while fluconazole was much less active. Because fluconazole was weakly active against these organisms in planktonic form, it is not surprising that biofilms were less susceptible to this antifungal compared with others included in the study. Observing SMIC80 results, it is apparent that the sessile form of C. auris CDC390 becomes particularly resistant to all of the antifungals. Table 3. Susceptibility profiles (mg/L) of sessile fungi (biofilm) to CSA-44, CSA-131 and three antifungal compounds   SMIC50 (SMIC80)   Antimicrobial  C. albicans  C. auris  C. auris  C. auris  C. auris  ATCC 90028  CDC381  CDC383  CDC386  CDC390  CSA-44  2.0 (8.0)  4.0 (16)  2.0 (8.0)  2.0 (4.0)  4.0 (64)  CSA-131  2.0 (4.0)  2.0 (32)  2.0 (4.0)  2.0 (4.0)  4.0 (64)  Amphotericin B  2.0 (8.0)  4.0 (16)  2.0 (4.0)  <1.0 (1.0)  8.0 (>64)  Caspofungin  2.0 (8.0)  4.0 (32)  8.0 (32)  8.0 (64)  8.0 (100)  Fluconazole  64 (200)  64 (>200)  64 (>200)  32 (200)  100 (>200)    SMIC50 (SMIC80)   Antimicrobial  C. albicans  C. auris  C. auris  C. auris  C. auris  ATCC 90028  CDC381  CDC383  CDC386  CDC390  CSA-44  2.0 (8.0)  4.0 (16)  2.0 (8.0)  2.0 (4.0)  4.0 (64)  CSA-131  2.0 (4.0)  2.0 (32)  2.0 (4.0)  2.0 (4.0)  4.0 (64)  Amphotericin B  2.0 (8.0)  4.0 (16)  2.0 (4.0)  <1.0 (1.0)  8.0 (>64)  Caspofungin  2.0 (8.0)  4.0 (32)  8.0 (32)  8.0 (64)  8.0 (100)  Fluconazole  64 (200)  64 (>200)  64 (>200)  32 (200)  100 (>200)  Table 3. Susceptibility profiles (mg/L) of sessile fungi (biofilm) to CSA-44, CSA-131 and three antifungal compounds   SMIC50 (SMIC80)   Antimicrobial  C. albicans  C. auris  C. auris  C. auris  C. auris  ATCC 90028  CDC381  CDC383  CDC386  CDC390  CSA-44  2.0 (8.0)  4.0 (16)  2.0 (8.0)  2.0 (4.0)  4.0 (64)  CSA-131  2.0 (4.0)  2.0 (32)  2.0 (4.0)  2.0 (4.0)  4.0 (64)  Amphotericin B  2.0 (8.0)  4.0 (16)  2.0 (4.0)  <1.0 (1.0)  8.0 (>64)  Caspofungin  2.0 (8.0)  4.0 (32)  8.0 (32)  8.0 (64)  8.0 (100)  Fluconazole  64 (200)  64 (>200)  64 (>200)  32 (200)  100 (>200)    SMIC50 (SMIC80)   Antimicrobial  C. albicans  C. auris  C. auris  C. auris  C. auris  ATCC 90028  CDC381  CDC383  CDC386  CDC390  CSA-44  2.0 (8.0)  4.0 (16)  2.0 (8.0)  2.0 (4.0)  4.0 (64)  CSA-131  2.0 (4.0)  2.0 (32)  2.0 (4.0)  2.0 (4.0)  4.0 (64)  Amphotericin B  2.0 (8.0)  4.0 (16)  2.0 (4.0)  <1.0 (1.0)  8.0 (>64)  Caspofungin  2.0 (8.0)  4.0 (32)  8.0 (32)  8.0 (64)  8.0 (100)  Fluconazole  64 (200)  64 (>200)  64 (>200)  32 (200)  100 (>200)  Confocal laser scanning microscopy of fungal biofilms To observe the antibiofilm properties of a lead ceragenin, biofilms of C. albicans and C. auris were treated with CSA-131, stained and imaged via confocal microscopy (Figure 3). Untreated biofilms exhibited expected aggregates of live cells, while treated biofilms showed comparable aggregates of dead cells. As with Gram-negative bacteria,49 the ceragenin was able to penetrate the extracellular matrix and exert antifungal activity without significantly compromising the biofilm morphology. The antibiofilm property of the ceragenins complements their ability to inhibit biofilm formation as reported in a recent study in which CSA-131, incorporated into a hydrogel, prevents biofilm formation on a medical device for extended periods.50 Figure 3. View largeDownload slide Confocal laser scanning micrographs (×60 magnification) of stained fungal biofilms. Green: live cells; red: dead cells. (a) C. albicans, untreated. (b) C. albicans, treated with CSA-131 (50 mg/L). (c) C. auris, untreated. (d) C. auris, treated with CSA-131 (50 mg/L). Figure 3. View largeDownload slide Confocal laser scanning micrographs (×60 magnification) of stained fungal biofilms. Green: live cells; red: dead cells. (a) C. albicans, untreated. (b) C. albicans, treated with CSA-131 (50 mg/L). (c) C. auris, untreated. (d) C. auris, treated with CSA-131 (50 mg/L). Antifungal activity of formulated ceragenins in tissue explants There are many potential applications of novel antifungal agents and some of the most widely useful would involve topical application to skin or mucosal tissues. For these types of applications, lead ceragenins would need to be formulated into gels or creams. HEC (typically used at 2.7% in water) can be used to form a lubricating gel into which antimicrobial compounds can be formulated. Pluronic® F-127 is a non-ionic surfactant that has shown compatibility with ceragenins with concomitant decreases in cytotoxic properties.51,52 Combining HEC, Pluronic® F-127 and either CSA-44 or CSA-131 at 0.5% (CSA-44L and CSA-131L) or 2.0% (CSA-44H and CSA-131H) gave stable, lubricious gels. These percentages of ceragenins are comparable to those of antifungals incorporated into commercially available antifungal products. Porcine vaginal mucosal explants were used to determine the antifungal activities of these gels. Nystatin (a polyene antifungal related to amphotericin B) was used as a comparator and the HEC/Pluronic® F-127 combination (vehicle) was used to observe effects of these compounds on the fungi. Fungal infection with either C. albicans or C. auris was established over 2 and 24 h of incubation, followed by treatment for 24 h with the test formulations. Over this time course, fungal populations grew to over 106 cfu/explant (Figure 4). In all experiments, the vehicle had no impact on fungal growth. Figure 4. View largeDownload slide Antifungal activities of nystatin (100 000 USP nystatin units) compared with HEC/Pluronic® F-127 formulations of CSA-44 and CSA-131 (CSA-44L and CSA-131L contain 0.5% ceragenin and CSA-44H and CSA-131H contain 2% ceragenin) in porcine vaginal mucosal tissue explants. Log10 cfu reduction from untreated growth control following 2 h (a and b) and 24 h (c and d) infections of C. auris or C. albicans on PVM. Data presented are means and SEMs. Analyses of variance (ANOVA) followed by Dunnett’s multiple comparison post-test were performed using the GraphPad PRISM software. *Significantly different from growth control (P < 0.5); #Significantly different from nystatin (P < 0.5). Figure 4. View largeDownload slide Antifungal activities of nystatin (100 000 USP nystatin units) compared with HEC/Pluronic® F-127 formulations of CSA-44 and CSA-131 (CSA-44L and CSA-131L contain 0.5% ceragenin and CSA-44H and CSA-131H contain 2% ceragenin) in porcine vaginal mucosal tissue explants. Log10 cfu reduction from untreated growth control following 2 h (a and b) and 24 h (c and d) infections of C. auris or C. albicans on PVM. Data presented are means and SEMs. Analyses of variance (ANOVA) followed by Dunnett’s multiple comparison post-test were performed using the GraphPad PRISM software. *Significantly different from growth control (P < 0.5); #Significantly different from nystatin (P < 0.5). At 2 h, nystatin decreased fungal counts with C. auris and C. albicans by ∼1 log, but only with C. albicans was this reduction statistically significant. In contrast, both ceragenins, at both concentrations, significantly reduced fungal counts at 2 h, with reductions as great as 5 logs (CSA-131H). At 24 h, nystatin-treated explants supported fungal counts comparable to controls with both species of fungi. At 24 h, the most potent activity of ceragenins was observed with C. albicans, with fungal counts significantly reduced relative to both control and nystatin-treated explants. With the highest concentrations of ceragenin (2%), fungal counts were reduced by >4 logs. Although the activity of the ceragenins at 24 h was not as dramatic with explants colonized with C. auris, significant reductions (∼3 logs) were observed with the highest concentration of the ceragenins. Significant reduction was also observed with the lower concentration (0.5%) of CSA-44. CSA-44 was also formulated into a cream and compared with a commercial minocycline product, Monistat 7, in the porcine vaginal mucosa (PVM) infection model. Based on efficacy of the ceragenins in the gel formulation, a concentration of 1% CSA-44 in a cream was used and compared with Monistat 7 (2% miconazole in cream formulation). No other formulations were evaluated in this assay. When tested for antifungal activity, the cream vehicle did not impact growth (i.e. growth of C. albicans in the presence of the cream formulation was not different from a blank control.) With C. albicans, Monistat 7 reduced fungal counts by a statistically significant amount at 2 h, compared with the growth control, but this effect was negligible at 24 h. With this organism, the CSA-44 cream formulation reduced counts by >3 logs at 2 h and by ∼2 logs at 24 h; both of these measurements were significantly different from both the vehicle and the Monistat 7-treated explants (Figure 5). Explants colonized with C. auris were not significantly affected by Monostat-7 treatment at 2 and 24 h. In contrast, the CSA-44-containing cream significantly reduced C. auris counts at both timepoints (>2 logs and >1 log reductions, respectively). Figure 5. View largeDownload slide Antifungal activities of Monistat 7 (2% miconazole) compared with a cream formulation of CSA-44 (1%) in porcine vaginal mucosal tissue explants. Log10 cfu reduction from untreated growth control following 2 h (a and b) and 24 h (c and d) infections of C. auris or C. albicans on PVM. Data presented are means and SEMs. Analyses of variance (ANOVA) followed by Dunnett’s multiple comparison post-test were performed using the GraphPad PRISM software. *Significantly different from growth control (P < 0.5); ^Significantly different from Monistat 7 (P < 0.5). Figure 5. View largeDownload slide Antifungal activities of Monistat 7 (2% miconazole) compared with a cream formulation of CSA-44 (1%) in porcine vaginal mucosal tissue explants. Log10 cfu reduction from untreated growth control following 2 h (a and b) and 24 h (c and d) infections of C. auris or C. albicans on PVM. Data presented are means and SEMs. Analyses of variance (ANOVA) followed by Dunnett’s multiple comparison post-test were performed using the GraphPad PRISM software. *Significantly different from growth control (P < 0.5); ^Significantly different from Monistat 7 (P < 0.5). Conclusions The spread of C. auris, the high mortality rates in patients with infection and the high prevalence of drug resistance among clinical isolates underscore the need for the development of novel antifungal agents. The mechanisms common to endogenous AMPs provide direction for investigation of therapeutic agents that mimic antimicrobial activities that nature has honed over millions of years. Due to their simple structures, ceragenins are attractive candidates for development of novel antifungal agents; they can be prepared on a large scale and they are stable in the presence of ubiquitous proteases. The ceragenins display antifungal activities with C. auris comparable to the most active agents tested and they do not show cross-resistance with other antifungals. The ceragenins also retain activity against sessile organisms in established biofilms. In formulated forms, ceragenins are active against high inocula of fungi in mucosal tissue. The substantial antifungal activities of these ceragenin formulations compared with nystatin and Monistat 7 highlight their potential for use as novel antifungals for topical or mucosal applications. Acknowledgements We thank N8 Medical, Inc., CSA Biotechnologies and Brigham Young University for generous funding. Funding This study was supported by funding from N8 Medical, Inc., CSA Biotechnologies and Brigham Young University. Transparency declarations P. B. S. is a paid consultant for N8 Medical, Inc. All other authors: none to declare. References 1 Lockhart SR, Etienne KA, Vallabhanemi S et al.   Simultaneous emergence of multidrug-resistant Candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses. Clin Infect Dis  2017; 64: 134– 40. Google Scholar CrossRef Search ADS PubMed  2 Clancy CJ, Nguyen MH. Emergence of Candida auris: an international call to arms. Clin Infect Dis  2017; 64: 141– 3. Google Scholar CrossRef Search ADS PubMed  3 Chowdhary A, Sharma C, Meis JF. Candida auris: a rapidly emerging cause of hospital-acquired multidrug-resistant fungal infections globally. PLoS Pathog  2017; 13: e1006290. Google Scholar CrossRef Search ADS PubMed  4 Todd B. Clinical alert: Candida auris. Am J Nurs  2017; 117: 53– 5. Google Scholar CrossRef Search ADS PubMed  5 Sarma S, Upadhyay S. Current perspective on emergence, diagnosis and drug resistance in Candida auris. Infect Drug Resist  2017; 10: 155– 65. Google Scholar CrossRef Search ADS PubMed  6 Tsay S, Welsh RM, Adams EH et al.   Ongoing transmission of Candida auris in health care facilities — United States, June 2016–May 2017. MMWR Morb Mortal Wkly Rep  2017; 66: 514– 5. Google Scholar CrossRef Search ADS PubMed  7 Mohsin J, Hagen F, Al-Balushi ZAM et al.   The first cases of Candida auris candidaemia in Oman. Mycoses  2017; 60: 569– 75. Google Scholar CrossRef Search ADS PubMed  8 Rudramurthy SM, Chakrabarti A, Paul RA et al.   Candida auris candidaemia in Indian ICUs: analysis of risk factors. J Antimicrob Chemother  2017; 72: 1794– 801. Google Scholar CrossRef Search ADS PubMed  9 Arendrup MC, Prakash A, Meletiadis J et al.   Comparison of EUCAST and CLSI reference microdilution MICs of eight antifungal compounds for Candida auris and associated epidemiological cutoff values. Antimicrob Agents Chemother  2017; 61: e00485– 17. Google Scholar PubMed  10 McCarthy MW, Walsh TJ. Drug development challenges and strategies to address emerging and resistant fungal pathogens. Exp Rev Anti-Infect Therapy  2017; 15: 577– 84. Google Scholar CrossRef Search ADS   11 Silva PM, Gonçalves S, Santos NC. Defensins: antifungal lessons from eukaryotes. Front Microbiol  2014; 5: 97. Google Scholar PubMed  12 Swidergall M, Ernst JF. Interplay between Candida albicans and the antimicrobial peptide armory. Eukaryot Cell  2014; 13: 950– 7. Google Scholar CrossRef Search ADS PubMed  13 Duncan VMS, O’Neil DA. Commercialization of antifungal peptides. Fungal Biol Rev  2013; 26: 156– 65. Google Scholar CrossRef Search ADS   14 Vriens K, Cammue BPA, Thevissen K. Antifungal plant defensins: mechanisms of action and production. Molecules  2014; 19: 12280– 303. Google Scholar CrossRef Search ADS PubMed  15 Hashemi MM, Holden BS, Durnas B et al.   Ceragenins as mimics of endogenous antimicrobial peptides. J Antimicrob Agents  2017; 3: 1000141. Google Scholar CrossRef Search ADS   16 Lai X-Z, Feng Y, Pollard J et al.   Ceragenins: cholic acid-based mimics of antimicrobial peptides. Acc Chem Res  2008; 41: 1233– 40. Google Scholar CrossRef Search ADS PubMed  17 Hashemi MM, Rovig J, Weber S et al.   Susceptibility of colistin-resistant, Gram-negative bacteria to antimicrobial peptides and ceragenins. Antimicrob Agents Chemother  2017; 61: e00292-17. Google Scholar CrossRef Search ADS PubMed  18 Vila-Farrés X, Callarisa AE, Gu X et al.   CSA-131, a ceragenin active against colistin-resistant Acinetobacter baumannii and Pseudomonas aeruginosa clinical isolates. Int J Antimicrob Agents  2015; 46: 568– 71. Google Scholar CrossRef Search ADS PubMed  19 Olekson MA, You T, Savage PB et al.   Antimicrobial ceragenins inhibit biofilms and affect mammalian cell viability and migration in vitro. FEBS Open Bio  2017; 7: 953– 67. Google Scholar CrossRef Search ADS PubMed  20 Durnas B, Wnorowska U, Pogoda K et al.   Candidacidal activity of selected ceragenins and human cathelicidin LL-37 in experimental settings mimicking infection sites. PLoS One  2016; 11: e0157242. Google Scholar CrossRef Search ADS PubMed  21 Kodedová M, Sychrová H. Synthetic antimicrobial peptides of the halcitines family disturb the membrane integrity of Candida cells. Biochim Biophys Acta  2017; 1859: 1851– 8. Google Scholar CrossRef Search ADS PubMed  22 Mangoni ML, Luca V, McDermott AM. Fighting microbial infections: a lesson from amphibian skin-derived esculentin-1 peptides. Peptides  2015; 71: 286– 95. Google Scholar CrossRef Search ADS PubMed  23 Tsai PW, Cheng YL, Hsieh WP et al.   Responses of Candida albicans to the human antimicrobial peptide LL-37. J Microbiol  2014; 52: 581– 9. Google Scholar CrossRef Search ADS PubMed  24 Lee H, Hwang JS, Lee DG. Scolopendin, an antimicrobial peptide from centipede, attenuates mitochondrial functions and triggers apoptosis in Candida albicans. Biochem J  2017; 474: 635– 45. Google Scholar CrossRef Search ADS PubMed  25 Lee J, Lee DG. Melittin triggers apoptosis in Candida albicans through the reactive oxygen species-mediated mitochondria/caspase-dependent pathway. FEMS Microbiol Lett  2014; 355: 36– 42. Google Scholar CrossRef Search ADS PubMed  26 Wong JH, Ng TB, Legowska A et al.   Antifungal action of human cathelicidin fragment (LL13-37) on Candida albicans. Peptides  2011; 32: 1996– 2002. Google Scholar CrossRef Search ADS PubMed  27 Park C, Lee DG. Melittin induces apoptotic features in Candida albicans. Biochem Biophys Res Commun  2010; 394: 170– 2. Google Scholar CrossRef Search ADS PubMed  28 Niemirowicz K, Durnaś B, Tokajuk G et al.   Formulation and candidacidal activity of magnetic nanoparticles coated with cathelicidin LL-37 and ceragenin CSA-13. Sci Rep  2017; 7: 4610. Google Scholar CrossRef Search ADS PubMed  29 Schindeler A, Yu NYC, Cheng TL et al.   Local delivery of the cationic steroid antibiotic CSA-90 enables osseous union in a rat open fracture model of Staphylococcus aureus infection. J Bone Joint Surg Am  2015; 97: 302– 9. Google Scholar CrossRef Search ADS PubMed  30 Bucki R, Niemirowicz K, Wnorowska U et al.   Bactericidal activity of ceragenin CSA-13 in cell culture and in an animal model of peritoneal infection. Antimicrob Agents Chemother  2015; 59: 6274– 82. Google Scholar CrossRef Search ADS PubMed  31 Sinclair CD, Pham TX, Williams DL et al.   Model development for determining the efficacy of a combination coating for the prevention of perioperative device related infections: a pilot study. J Biomed Mater Res B Appl Biomater  2013; 101: 1143– 53. Google Scholar CrossRef Search ADS PubMed  32 Williams DL, Haymond BS, Beck PJ et al.   In vivo efficacy of a silicone-cationic steroid antimicrobial coating to prevent implant-related infection. Biomaterials  2012; 33: 8641– 56. Google Scholar CrossRef Search ADS PubMed  33 Ding B, Yin N, Liu Y et al.   Origins of cell selectivity of cationic steroid antibiotics. J Am Chem Soc  2004; 126: 13642– 8. Google Scholar CrossRef Search ADS PubMed  34 Guan Q, Schmidt EJ, Boswell SR et al.   Preparation and characterization of cholic acid-derived antimicrobial agents with controlled stabilities. Org Lett  2000; 2: 2837– 40. Google Scholar CrossRef Search ADS PubMed  35 Boikov DA, Locke JB, James KD et al.   In vitro activity of the novel echinocandin CD101 at pH 7 and 4 against Candida spp. isolates from patients with vulvovaginal candidiasis. J Antimicrob Chemother  2017; 72: 1355– 8. Google Scholar CrossRef Search ADS PubMed  36 De Seta F, Schmidt M, Vu B et al.   Antifungal mechanisms supporting boric acid therapy of Candida vaginitis. J Antimicrob Chemother  2009; 63: 325– 36. Google Scholar CrossRef Search ADS PubMed  37 Valentín A, Cantón E, Pemán J et al.   In vitro activity of anidulafungin in combination with amphotericin B or voriconazole against biofilms of five Candida species. J Antimicrob Chemother  2016; 71: 3449– 52. Google Scholar CrossRef Search ADS PubMed  38 Zhang Y, Chen Y-Y, Huang L et al.   The antifungal effects and mechanical properties of silver bromide/cationic polymer nano-composite-modified Poly-methyl methacrylate-based dental resin. Sci Rep  2017; 7: 1547. Google Scholar CrossRef Search ADS PubMed  39 Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Third Informational Supplement M27-A3 . CLSI, Wayne, PA, USA, 2008. 40 Sóczó G, Kardos G, McNicholas PM et al.   Correlation of posaconazole minimum fungicidal concentration and time–kill test against nine Candida species. J Antimicrob Chemother  2007; 60: 1004– 9. Google Scholar CrossRef Search ADS PubMed  41 Moss BJ, Kim Y, Nandakumar MP et al.   Quantifying metabolic activity of filamentous fungi using a colorimetric XTT assay. Biotechnol Prog  2008; 24: 780– 3. Google Scholar CrossRef Search ADS PubMed  42 Anderson MJ, Parks PJ, Peterson ML. A mucosal model to study microbial biofilm development and anti-biofilm therapeutics. J Microbiol Methods  2013; 92: 201– 8. Google Scholar CrossRef Search ADS PubMed  43 Bruzual I, Riggle P, Hadley S et al.   Biofilm formation by fluconazole-resistant Candida albicans strains is inhibited by fluconazole. J Antimicrob Chemother  2007; 59: 441– 50. Google Scholar CrossRef Search ADS PubMed  44 Kagan S, Jabbour A, Sionov E et al.   Anti-Candida albicans biofilm effect of novel heterocyclic compounds. J Antimicrob Chemother  2014; 69: 416– 27. Google Scholar CrossRef Search ADS PubMed  45 Khan MSA, Ahmad I. Antibiofilm activity of certain phytocompounds and their synergy with fluconazole against Candida albicans biofilms. J Antimicrob Chemother  2012; 67: 618– 21. Google Scholar CrossRef Search ADS PubMed  46 Lazzell AL, Chaturvedi AK, Pierce CG et al.   Treatment and prevention of Candida albicans biofilms with caspofungin in a novel central venous catheter murine model of candidiasis. J Antimicrob Chemother  2009; 64: 567– 70. Google Scholar CrossRef Search ADS PubMed  47 Larkin E, Hager C, Chandra J et al.   The emerging Candida auris: characterization of growth phenotype, virulence factors, antifungal activity, and effect of SCY-078, a novel glucan synthesis inhibitor, on growth morphology and biofilm formation. Antimicrob Agents Chemother  2017; 61: e02396– 16. Google Scholar CrossRef Search ADS PubMed  48 Oh BJ, Shin JH, Kim M-N et al.   Biofilm formation and genotyping of Candida haemulonii, Candida pseudohaemulonii, and a proposed new species (Candida auris) isolates from Korea. Med Mycol  2011; 49: 98– 102. Google Scholar CrossRef Search ADS PubMed  49 Nagant C, Pitts B, Stewart PS et al.   Study of the effect of antimicrobial peptide mimic, CSA-13, on an established biofilm formed by Pseudomonas aeruginosa. Microbiol Open  2013; 2: 318– 25. Google Scholar CrossRef Search ADS   50 Hashemi MM, Rovig J, Bateman J et al.   Preclinical testing of a broad-spectrum antimicrobial endotracheal tube coated with an innate immune synthetic mimic. J Antimicrob Chemother  2018; 73: 143– 50. Google Scholar CrossRef Search ADS PubMed  51 Nagant C, Savage PB, Dehaye JP. Effect of pluronic acid F-127 on the toxicity towards eukaryotic cells of CSA-13, a cationic steroid analogue of antimicrobial peptides. J Appl Microbiol  2012; 112: 1173– 83. Google Scholar CrossRef Search ADS PubMed  52 Leszczyńska K, Namiot A, Cruz K et al.   Potential of ceragenin CSA-13 and its mixture with pluronic F-127 as treatment of topical bacterial infections. J Appl Microbiol  2011; 110: 229– 38. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

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Abstract

Abstract Background Candida auris has emerged as a serious threat to human health. Of particular concern are the resistance profiles of many clinical isolates, with some being resistant to multiple classes of antifungals. Objectives Measure susceptibilities of C. auris isolates, in planktonic and biofilm forms, to ceragenins (CSAs). Determine the effectiveness of selected ceragenins in gel and cream formulations in eradicating fungal infections in tissue explants. Materials and methods A collection of 100 C. auris isolates available at CDC was screened for susceptibility to a lead ceragenin. A smaller collection was used to characterize antifungal activities of other ceragenins against organisms in planktonic and biofilm forms. Effects of ceragenins on fungal cells and biofilms were observed via microscopy. An ex vivo model of mucosal fungal infection was used to evaluate formulated forms of lead ceragenins. Results Lead ceragenins displayed activities comparable to those of known antifungal agents against C. auris isolates with MICs of 0.5–8 mg/L and minimum fungicidal concentrations (MFCs) of 2–64 mg/L. No cross-resistance with other antifungals was observed. Fungal cell morphology was altered in response to ceragenin treatment. Ceragenins exhibited activity against sessile organisms in biofilms. Gel and cream formulations including 2% CSA-44 or CSA-131 resulted in reductions of over 4 logs against established fungal infections in ex vivo mucosal tissues. Conclusions Ceragenins demonstrated activity against C. auris, suggesting that these compounds warrant further study to determine whether they can be used for topical applications to skin and mucosal tissues for treatment of infections with C. auris and other fungi. Introduction Candida auris has emerged as a global threat; since the initial report from Japan in 2009, it has spread and been isolated on five continents.1–6,C. auris can be a substantial cause of nosocomial infections in some settings and is associated with high levels of mortality.7,8 Of particular concern is the drug resistance found among many isolates. Resistance to azole antifungal agents with clinical isolates is common and resistance of some strains of C. auris to all three classes of commonly used antifungals (azoles, polyenes and echinocandins) has been observed.1,9 The emergence of this pathogen has led to efforts to better track and characterize infections1,6 and it provides a strong impetus for the development of novel antifungal agents active against drug-resistant organisms.10 Higher organisms have faced threats from fungal pathogens for hundreds of millions of years and evolutionary pressures have yielded antifungal innate immune functions effective against organisms in both planktonic and biofilm forms. Antimicrobial peptides (AMPs) constitute a key component of these innate immune defences and AMPs from a variety of sources have shown potent antifungal activities.11–14 The prevalence of AMPs, in organisms ranging from mammals to amphibians to insects to plants, suggests that AMPs retain antimicrobial activities over extended periods without generation of widespread resistance. Recognizing the antifungal activities of AMPs and their anticipated retention of potency with extensive use, substantial efforts have been made to develop antifungal AMPs for clinical use.13 Impediments to clinical use include the relatively high cost of large-scale preparation of peptide therapeutic agents compared with that of small molecules and the instability of linear peptides in the presence of ubiquitous proteases. In an effort to overcome the challenges associated with these peptide therapeutic agents, while retaining the same mechanisms of antimicrobial action, we have developed a class of non-peptide molecules, based on a common bile acid, that mimic the amphiphilic morphology common to AMPs.15,16 Molecules of this class, termed ceragenins (CSAs) (examples shown in Figure 1), can be prepared on a large scale and, because they are not peptide-based, ceragenins are not substrates for proteases. Lead ceragenins are broad-spectrum antimicrobials with activity against drug-resistant bacteria, including colistin-resistant Gram-negative bacteria.17,18 Ceragenins also mimic ‘secondary’ activities of AMPs associated with wound healing.19 Figure 1. View largeDownload slide Structures of selected ceragenins. Figure 1. View largeDownload slide Structures of selected ceragenins. Recently, antifungal activities of selected ceragenins were reported,20 with MICs of ceragenins comparable to or below those measured with amphotericin B, fluconazole and AMPs, omiganan and LL-37, for multiple strains of Candida albicans. Fungi in the genera of Paecilomyces, Cryptococcus, Aspergillus, Scedosporium, Rhizopus, Blastomyces and Apophysomyces also proved susceptible to the ceragenins tested.20 Atomic force microscopy images of C. albicans untreated and treated with ceragenin CSA-13 showed changes in surface morphology of cells treated with the ceragenin, which were interpreted as showing membrane activity.20 Studies of mechanisms of antifungal activities of AMPs have concluded that membrane perturbation plays a key role21–23 and increases in damage by reactive oxygen species (ROS) and attenuation of mitochondrial functions, leading to apoptosis, have been observed.24–27 In a recent study, magnetic nanoparticles adorned with either the AMP LL-37 or CSA-13 resulted in generation of ROS in Candida spp.28 Considering the need for novel antifungal agents in the face of the growing threat of C. auris infection, our objectives in the studies described herein were to determine the antifungal activity of selected ceragenins against isolates of C. auris collected by CDC and to compare these activities with those of representatives of the three major classes of antifungal agents. Activities measured included both MICs and minimum fungicidal concentrations (MFCs) on planktonic organisms. Because fungi may grow in sessile form in biofilms, we also measured the activities of selected ceragenins against established biofilm forms of C. auris. To better understand the antifungal activities of ceragenins, scanning electron microscopy (SEM) images of C. auris, with and without ceragenin treatment, were acquired. Although ceragenins are well-tolerated in a variety of routes of administration,29–32 topical application to skin or mucosal tissues to treat fungal infections represents an attractive use of the antimicrobials. To determine the potential for use in this venue, different formulations of ceragenins CSA-44 and CSA-131 were evaluated in an ex vivo porcine vaginal mucosal tissue model infected with C. albicans or C. auris and compared with nystatin or a commercial cream containing miconazole. Materials and methods Materials and fungal strains CDC (Atlanta, GA, USA) has catalogued over 100 clinical isolates of C. auris for use in determining susceptibility patterns and these were used in initial studies with ceragenin CSA-131. These isolates represent all four of the known clades of C. auris and originated from various countries. These isolates were characterized as susceptible or resistant to fluconazole and echinocandins based on the MICs published by CDC (https://www.cdc.gov/fungal/diseases/candidiasis/recommendations.html), although breakpoints specific for C. auris have not yet been established. Ten additional isolates of C. auris and one strain of C. albicans (ATCC 90028, Manassas, VA, USA) were used to further characterize susceptibility to other ceragenins. Ceragenins CSA-44, CSA-131, CSA-142 and CSA-144 were prepared as reported previously.33,34 Three antifungal drugs belonging to the three main classes of commonly used antifungal compounds were used in comparison: amphotericin B (Fisher Scientific, Pittsburgh, PA, USA), fluconazole and caspofungin (Sigma–Aldrich, St Louis, MO, USA). The culture media used were Sabouraud dextrose broth (SDB) or agar (SDA) and Roswell Park Memorial Institute medium 1640 (RPMI) (Sigma–Aldrich) buffered at pH 7.0 with 165 mM morpholinopropanesulfonic acid (MOPS) (Sigma–Aldrich). Published protocols for growth of Candida spp. have used either SDB or RPMI,35–38 and to observe any medium-dependent changes in susceptibility, MICs and MFCs were measured in both media. Determination of susceptibility profiles of planktonic fungi MICs of the C. auris strains were measured according to the broth microdilution protocol from the CLSI M27-A3 document.39 With the entire CDC collection, RPMI medium was used in the measurement; inocula of 0.5 × 103–2.5 × 103 cfu/mL were prepared and used to seed pre-prepared drug plates containing CSA-131. These plates were incubated at 35°C and read after 24 h. With the smaller collection (10 isolates), two media (SDB and RPMI) were used to evaluate the antifungal activity of all four classes of antifungal compounds used in this study. MFCs were determined by plating 10 μL from each well of the MIC plate (after 24 h incubation) on SDA plates and measuring the lowest concentration of the antibiotic which eliminated 99.9% (3 logs reduction) of the colonies formed on the plates after 48 h of incubation at 35°C.40 Determination of susceptibility profiles of fungal biofilms To quantify the biofilm formed by clinical isolates of C. auris and determine the sessile susceptibility profile of ceragenins, metabolic activity within biofilms was imaged using XTT as described by Moss et al.41 In this assay, metabolic activity of cells in the biofilm is measured based on their reduction of XTT. Initially, biofilms were formed in sterilized polystyrene flat-bottomed 96-well microtitre plates (Sarstedt, USA) incubated at 35°C for 48 h and washed three times with PBS (Sigma–Aldrich) to remove planktonic cells. Ceragenins were then added to the wells in a concentration range of 1–128 mg/L and incubated for 24 h. A 10 mM menadione (Sigma–Aldrich) solution was prepared in 100% acetone and added to an XTT solution (0.5 mg/mL) to achieve a final concentration of 1 μM menadione. Aliquots of the XTT/menadione solution (100 μL) were added to each well containing biofilm and negative control wells. Plates were wrapped in aluminium foil and incubated at 37°C for 2 h. Aliquots of the supernatant (70 μL) were removed from each well and the OD was measured at 490 nm using a microtitre plate reader. Corresponding OD values for each strain were subtracted from the negative control values to calculate sessile MICs (SMICs). The SMIC50 and SMIC80 represent the concentration of the drug where the absorbance of the biofilm decreased by 50% or by 80%, respectively, compared with the biofilm formed by the same strain in the absence of the drug. Results are from three independent experiments. SEM of C. auris To observe the effect of ceragenins on cell membranes, C. auris CDC390 was cultured to mid-log phase and washed three times with PBS. Fungi were resuspended in PBS (OD600 = 0.2). CSA-131 (25 or 50 mg/L) was added and the mixtures were incubated at 37°C for 1 h. A control was prepared by incubating the fungal suspension without adding CSA-131. After collection via centrifugation, cells were washed with PBS three times. Glutaraldehyde (2.5%) in PBS was added to fix the cells at 4°C overnight. Resulting material was washed five times with PBS at 5000 rpm for 10 min using a microhaematocrit centrifuge (Hettich Mikro 20, Hettich, Tuttlingen, Germany) to remove the glutaraldehyde. Osmium tetroxide (0.5 mL) was used as a second fixative reagent and samples were stored at room temperature for 2–3 h. Cells were washed with PBS five times at 14 000 rpm for 8 min. A graded ethanol series (10%–100%) was used to dehydrate the cells. Samples were collected by centrifugation and the supernatant was discarded after each cycle. Finally, the samples were exposed to air in a desiccator at room temperature for 24 h. Dried fungal specimens were sputter-coated with 5–10 nm of a gold/palladium alloy and visualized under a scanning electron microscope (FEI Helios NanoLab 600 SEM/FIB, Hillsboro, OR, USA). Confocal laser scanning microscopy To observe the effects of treatment of fungal biofilms with CSA-131, aliquots (300 μL) of C. albicans and C. auris CDC383 (106 cfu/mL) were placed into separate single wells in a Lab-Tek® Chamber Slide™ (Nunc, Inc., Naperville, IL, USA) and incubated for 48 h. The biofilms were treated with CSA-131 (50 μL of a 50 mg/L solution) and incubated for 24 h at 35°C. After incubation, the solution was carefully removed and the wells were washed three times with PBS. Biofilms were stained with BacLight Live/Dead Viability Kit (L13152, Molecular Probes, Inc) according to the manufacturer’s instructions and observed at ×60 magnification using a confocal laser scanning microscope (Olympus FLUOVIEW FV1000). Assays in ex vivo tissue Ex vivo experiments were performed using porcine vaginal mucosal explants as previously described.42 Normal healthy porcine vaginal tissue was excised from animals at slaughter (Theurer’s Quality Meats, Lewiston, UT, USA) and transported to the laboratory on ice. Tissue was trimmed and collected in RPMI-1640 medium supplemented with penicillin (50 IU/mL, MP Biomedicals, Solon, OH, USA) and streptomycin (50 IU/mL, MP Biomedicals). Antibiotics were included to decolonize normal flora, which may interfere with biofilm formation. Explants of uniform size were obtained using a 5 mm biopsy punch. Excess muscle was trimmed away with a scalpel. After antibiotic washout (three changes of RPMI-1640 medium followed by 30 min incubation at 37°C), explants were transferred mucosal side up onto a polyethylene terephthalate 0.4 μm cell culture insert (BD Falcon, Franklin Lakes, NJ, USA) and inoculated with 2 μL (105 cfu/explant) of C. auris (CDC390) or C. albicans (ATCC 90028) prepared from overnight cultures in Todd Hewitt broth. Following 2 and 24 h incubation at 37°C, explants (n = 3) were treated with CSA-44L or CSA-131L (100 μL, 0.5% active) or CSA-44H and CSA-131H (100 μL, 2.0% active) in a hydroxyethylcellulose (HEC) gel formulation and subsequently incubated for 24 h at 37°C. For studies of cream formulations, aliquots of the test articles (100 μL) were spread evenly over explants. After treatment, tissues were suspended in a neutralizing broth [250 μL, Dey/Engley (HiMedia Laboratories, West Chester, PA, USA)], vortex-mixed (highest setting for 4 min) and then serially diluted in PBS and plated. Samples were spread on tryptic soy agar containing 5% sheep blood (BD Falcon) using a spiral plater (Microbiology International, Frederick, MD, USA) then incubated for 24 h at 37°C. The cream vehicle was assayed for antifungal activity by placing aliquots (100 μL) in a 96-well plate and adding fungal inocula (C. albicans ATCC 90028, 100 μL of 103, 104 or 105 cfu/mL cultures). Resulting samples were incubated (37°C) for 24 h. Aliquots from each sample were plated, incubated for 48 h and counted. Results were compared with controls that were not treated with the cream vehicle. Statistical analysis Analyses of variance (ANOVA) were performed by Dunnett’s multiple comparison post-test using the GraphPad PRISM software (GraphPad Software, Inc., La Jolla, CA, USA). Results and discussion Susceptibility of planktonic fungi to ceragenins The susceptibilities of 100 clinical isolates to a lead ceragenin, CSA-131, were determined and the distribution of CSA-131 MIC values ranged from 0.5 to 1 mg/L (Table 1). The overall mode was 1 mg/L and both the MIC50 and the MIC90 were 1 mg/L. CSA-131 showed activity against all four clades of C. auris with no variation in activity between the clades. There was no loss in activity for those isolates that were previously determined to be resistant to fluconazole and/or an echinocandin. Table 1. MICs of CSA-131 for fluconazole-resistant, fluconazole-susceptible and echinocandin-resistant C. auris isolates     MIC (mg/L)   C. auris isolate  n  range  mode  MIC50  MIC90  Fluconazole-susceptible  30  0.5–1.0  NA  0.5  1.0  Fluconazole-resistant  70  0.5–1.0  1.0  1.0  1.0  Echinocandin-resistant  7  0.5–1.0  1.0  1.0  1.0      MIC (mg/L)   C. auris isolate  n  range  mode  MIC50  MIC90  Fluconazole-susceptible  30  0.5–1.0  NA  0.5  1.0  Fluconazole-resistant  70  0.5–1.0  1.0  1.0  1.0  Echinocandin-resistant  7  0.5–1.0  1.0  1.0  1.0  NA, not applicable (bimodal with equal numbers of MICs of 0.5 and 1.0 mg/L). Table 1. MICs of CSA-131 for fluconazole-resistant, fluconazole-susceptible and echinocandin-resistant C. auris isolates     MIC (mg/L)   C. auris isolate  n  range  mode  MIC50  MIC90  Fluconazole-susceptible  30  0.5–1.0  NA  0.5  1.0  Fluconazole-resistant  70  0.5–1.0  1.0  1.0  1.0  Echinocandin-resistant  7  0.5–1.0  1.0  1.0  1.0      MIC (mg/L)   C. auris isolate  n  range  mode  MIC50  MIC90  Fluconazole-susceptible  30  0.5–1.0  NA  0.5  1.0  Fluconazole-resistant  70  0.5–1.0  1.0  1.0  1.0  Echinocandin-resistant  7  0.5–1.0  1.0  1.0  1.0  NA, not applicable (bimodal with equal numbers of MICs of 0.5 and 1.0 mg/L). To verify that other lead ceragenins also demonstrated activity against C. auris, studies were performed with a smaller collection of isolates and activities were compared with those of a strain of C. albicans. Both MICs and MFCs were measured in SDB or RPMI of CSA-44, CSA-131, CSA-142, CSA-144 and representatives of the three major classes of antifungal agents. As observed with the larger collection of C. auris isolates, MICs of CSA-131 were either 0.5 or 1.0 mg/L and MICs of CSA-44 and CSA-144 were similar (Table 2). MICs of CSA-142 were relatively higher. The strains tested were associated with relatively high MICs of fluconazole and one of the strains (CDC383) showed resistance to caspofungin. Compared with caspofungin, amphotericin B and fluconazole, the ceragenins gave lower MFCs, suggesting greater fungicidal activity compared with these other antifungal agents. Among the representative antifungal agents, caspofungin is the most closely related to ceragenins; it is a cationic molecule appended with a lipid chain. Similarly, ceragenins are cationic and a lipid chain is required for activity. Nevertheless, echinocandin-resistant C. auris strains (CDC383 for example) were fully susceptible to ceragenins (Table 1). In general, the medium in which MICs and MFCs were determined did not play a substantial role; MICs of the majority of the antifungal agents were identical in both media, as were MFCs. Table 2. Comparison of the susceptibility of clinical isolates of C. auris to selected ceragenins and three major classes of antifungal agents in SDB and RPMI   MIC (MFC) mg/L   Strains  CSA-44  CSA-131  CSA-142  CSA-144  CPF  AMB  FLC  SDB  SDB  SDB  SDB  SDB  SDB  SDB  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  Candida auris   CDC381  0.5 (2.0)a  0.5 (8.0)a  4.0 (32)a  0.5 (2.0)  2.0 (64)a  1.0 (48)  16 (>100)  [1.0 (8.0)]  [1.0 (64)]  [32 (>100)]   CDC382  0.5 (4.0)a  0.5 (8.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC383  0.5 (8.0)  1.0 (10)  2.0 (64)a  1.0 (8)a  32 (64)  1.0 (32)  64(>100)  [0.5 (16)]  [0.5 (8.0)]  [16 (48)]  [1.0 (64)]  [>100 (>100)]   CDC384  0.5 (4.0)a  0.5 (4.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC385  0.5 (16)a  0.5 (4.0)a  8.0 (32)a  1.0 (8.0)a  NM  NM  NM   CDC386  0.5 (8.0)a  1.0 (8.0)  4.0 (32)a  0.5 (8.0)  2.0 (64)a  2.0 (48)  64(>100)  [0.5 (8.0)]  [1.0 (16)]  [1.0 (64)]  [>100(>100)]   CDC387  0.5 (8.0)a  0.5 (8.0)a  4.0 (32)a  0.5 (8)a  NM  NM  NM   CDC388  1.0 (8.0)a  0.5 (4.0)a  4.0 (24)a  2.0 (8.0)a  NM  NM  NM   CDC389  0.5 (8.0)a  0.5 (8.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC390  0.5 (8.0)  0.5 (4.0)  4.0 (16)a  1.0 (8.0)a  2.0 (100)a  4.0(64)a  64 (>100)  [1.0 (32)]  [0.5 (8.0)]  [>100 (>100)]  Candida albicans   ATCC 90028  0.5 (8.0)  0.5 (4.0)  2.0 (8.0)a  2.0 (8.0)a  2.0 (32)  2.0 (100)  24 (>100)  [0.5 (16)]  [0.5 (8.0)]  [1.0 (8.0)]  [2.0 (64)]  [32 (>100)]    MIC (MFC) mg/L   Strains  CSA-44  CSA-131  CSA-142  CSA-144  CPF  AMB  FLC  SDB  SDB  SDB  SDB  SDB  SDB  SDB  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  Candida auris   CDC381  0.5 (2.0)a  0.5 (8.0)a  4.0 (32)a  0.5 (2.0)  2.0 (64)a  1.0 (48)  16 (>100)  [1.0 (8.0)]  [1.0 (64)]  [32 (>100)]   CDC382  0.5 (4.0)a  0.5 (8.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC383  0.5 (8.0)  1.0 (10)  2.0 (64)a  1.0 (8)a  32 (64)  1.0 (32)  64(>100)  [0.5 (16)]  [0.5 (8.0)]  [16 (48)]  [1.0 (64)]  [>100 (>100)]   CDC384  0.5 (4.0)a  0.5 (4.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC385  0.5 (16)a  0.5 (4.0)a  8.0 (32)a  1.0 (8.0)a  NM  NM  NM   CDC386  0.5 (8.0)a  1.0 (8.0)  4.0 (32)a  0.5 (8.0)  2.0 (64)a  2.0 (48)  64(>100)  [0.5 (8.0)]  [1.0 (16)]  [1.0 (64)]  [>100(>100)]   CDC387  0.5 (8.0)a  0.5 (8.0)a  4.0 (32)a  0.5 (8)a  NM  NM  NM   CDC388  1.0 (8.0)a  0.5 (4.0)a  4.0 (24)a  2.0 (8.0)a  NM  NM  NM   CDC389  0.5 (8.0)a  0.5 (8.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC390  0.5 (8.0)  0.5 (4.0)  4.0 (16)a  1.0 (8.0)a  2.0 (100)a  4.0(64)a  64 (>100)  [1.0 (32)]  [0.5 (8.0)]  [>100 (>100)]  Candida albicans   ATCC 90028  0.5 (8.0)  0.5 (4.0)  2.0 (8.0)a  2.0 (8.0)a  2.0 (32)  2.0 (100)  24 (>100)  [0.5 (16)]  [0.5 (8.0)]  [1.0 (8.0)]  [2.0 (64)]  [32 (>100)]  CAS, caspofungin; AMB, amphotericin B; FLC, fluconazole; NM, not measured. a Same result in both media. Table 2. Comparison of the susceptibility of clinical isolates of C. auris to selected ceragenins and three major classes of antifungal agents in SDB and RPMI   MIC (MFC) mg/L   Strains  CSA-44  CSA-131  CSA-142  CSA-144  CPF  AMB  FLC  SDB  SDB  SDB  SDB  SDB  SDB  SDB  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  Candida auris   CDC381  0.5 (2.0)a  0.5 (8.0)a  4.0 (32)a  0.5 (2.0)  2.0 (64)a  1.0 (48)  16 (>100)  [1.0 (8.0)]  [1.0 (64)]  [32 (>100)]   CDC382  0.5 (4.0)a  0.5 (8.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC383  0.5 (8.0)  1.0 (10)  2.0 (64)a  1.0 (8)a  32 (64)  1.0 (32)  64(>100)  [0.5 (16)]  [0.5 (8.0)]  [16 (48)]  [1.0 (64)]  [>100 (>100)]   CDC384  0.5 (4.0)a  0.5 (4.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC385  0.5 (16)a  0.5 (4.0)a  8.0 (32)a  1.0 (8.0)a  NM  NM  NM   CDC386  0.5 (8.0)a  1.0 (8.0)  4.0 (32)a  0.5 (8.0)  2.0 (64)a  2.0 (48)  64(>100)  [0.5 (8.0)]  [1.0 (16)]  [1.0 (64)]  [>100(>100)]   CDC387  0.5 (8.0)a  0.5 (8.0)a  4.0 (32)a  0.5 (8)a  NM  NM  NM   CDC388  1.0 (8.0)a  0.5 (4.0)a  4.0 (24)a  2.0 (8.0)a  NM  NM  NM   CDC389  0.5 (8.0)a  0.5 (8.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC390  0.5 (8.0)  0.5 (4.0)  4.0 (16)a  1.0 (8.0)a  2.0 (100)a  4.0(64)a  64 (>100)  [1.0 (32)]  [0.5 (8.0)]  [>100 (>100)]  Candida albicans   ATCC 90028  0.5 (8.0)  0.5 (4.0)  2.0 (8.0)a  2.0 (8.0)a  2.0 (32)  2.0 (100)  24 (>100)  [0.5 (16)]  [0.5 (8.0)]  [1.0 (8.0)]  [2.0 (64)]  [32 (>100)]    MIC (MFC) mg/L   Strains  CSA-44  CSA-131  CSA-142  CSA-144  CPF  AMB  FLC  SDB  SDB  SDB  SDB  SDB  SDB  SDB  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  [RPMI]  Candida auris   CDC381  0.5 (2.0)a  0.5 (8.0)a  4.0 (32)a  0.5 (2.0)  2.0 (64)a  1.0 (48)  16 (>100)  [1.0 (8.0)]  [1.0 (64)]  [32 (>100)]   CDC382  0.5 (4.0)a  0.5 (8.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC383  0.5 (8.0)  1.0 (10)  2.0 (64)a  1.0 (8)a  32 (64)  1.0 (32)  64(>100)  [0.5 (16)]  [0.5 (8.0)]  [16 (48)]  [1.0 (64)]  [>100 (>100)]   CDC384  0.5 (4.0)a  0.5 (4.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC385  0.5 (16)a  0.5 (4.0)a  8.0 (32)a  1.0 (8.0)a  NM  NM  NM   CDC386  0.5 (8.0)a  1.0 (8.0)  4.0 (32)a  0.5 (8.0)  2.0 (64)a  2.0 (48)  64(>100)  [0.5 (8.0)]  [1.0 (16)]  [1.0 (64)]  [>100(>100)]   CDC387  0.5 (8.0)a  0.5 (8.0)a  4.0 (32)a  0.5 (8)a  NM  NM  NM   CDC388  1.0 (8.0)a  0.5 (4.0)a  4.0 (24)a  2.0 (8.0)a  NM  NM  NM   CDC389  0.5 (8.0)a  0.5 (8.0)a  4.0 (24)a  1.0 (8.0)a  NM  NM  NM   CDC390  0.5 (8.0)  0.5 (4.0)  4.0 (16)a  1.0 (8.0)a  2.0 (100)a  4.0(64)a  64 (>100)  [1.0 (32)]  [0.5 (8.0)]  [>100 (>100)]  Candida albicans   ATCC 90028  0.5 (8.0)  0.5 (4.0)  2.0 (8.0)a  2.0 (8.0)a  2.0 (32)  2.0 (100)  24 (>100)  [0.5 (16)]  [0.5 (8.0)]  [1.0 (8.0)]  [2.0 (64)]  [32 (>100)]  CAS, caspofungin; AMB, amphotericin B; FLC, fluconazole; NM, not measured. a Same result in both media. SEM of ceragenin-treated fungi As described above, membrane disruption has been identified as a major mechanism of antifungal activity of AMPs,21–23 among other possible mechanisms. Changes in cell morphology upon treatment with AMPs have been attributed to membrane interactions; these include loss of smooth cell surfaces with marked invagination at high AMP concentrations. Similar morphological changes in C. albicans were observed with the human AMP, LL-37, and the ceragenin, CSA-13.20 The impacts of CSA-131 on the morphology of C. auris are shown in Figure 2. As with C. albicans treated with LL-37 or CSA-13, CSA-131 alters cell shape; at 50 mg/L, CSA-131 causes cells to buckle in upon themselves and some cells appear to have merged. These changes in cell morphology were observed at concentrations well above MFCs and antifungal activity may be due to more subtle changes in cell permeability at lower concentrations of the ceragenin. At MFCs, little or no morphological changes in fungal cells were apparent. Figure 2. View largeDownload slide Scanning electron photomicrograph of untreated C. auris CDC390 (a and b) and treated with 25 mg/L CSA-131 (c and d) and 50 mg/L CSA-131 (e and f). Figure 2. View largeDownload slide Scanning electron photomicrograph of untreated C. auris CDC390 (a and b) and treated with 25 mg/L CSA-131 (c and d) and 50 mg/L CSA-131 (e and f). Susceptibility of fungal biofilms to ceragenins The abilities of bacteria and fungi to form biofilms, in which sessile organisms reside, contribute to drug resistance and protection of persister organisms that provide sources for chronic infections. The abilities of C. albicans to form biofilms are well-established;43–46 however, differing results for biofilm-forming propensities of C. auris have been reported.47,48 Using a measure of metabolic activity, we compared biofilm-forming properties of a C. albicans reference strain with those of four isolates of C. auris (CDC381, CDC383, CDC386 and CDC390). Biofilms were grown under identical conditions and, as reported,47C. auris strains were less efficient than the C. albicans strain at biofilm formation. Using the XTT-based assay, metabolic activity of the C. auris biofilms was measured as approximately 50% of that of C. albicans (data not shown). Having established the biofilm-forming characteristics of C. auris isolates, we used metabolic activity of biofilms to determine the activities of representative ceragenins and antifungals against sessile organisms making up these biofilms. Concentrations necessary to reduce biofilms by 50% and 80% are shown in Table 3. The ceragenins, amphotericin B and caspofungin demonstrated strong activity against the biofilms, while fluconazole was much less active. Because fluconazole was weakly active against these organisms in planktonic form, it is not surprising that biofilms were less susceptible to this antifungal compared with others included in the study. Observing SMIC80 results, it is apparent that the sessile form of C. auris CDC390 becomes particularly resistant to all of the antifungals. Table 3. Susceptibility profiles (mg/L) of sessile fungi (biofilm) to CSA-44, CSA-131 and three antifungal compounds   SMIC50 (SMIC80)   Antimicrobial  C. albicans  C. auris  C. auris  C. auris  C. auris  ATCC 90028  CDC381  CDC383  CDC386  CDC390  CSA-44  2.0 (8.0)  4.0 (16)  2.0 (8.0)  2.0 (4.0)  4.0 (64)  CSA-131  2.0 (4.0)  2.0 (32)  2.0 (4.0)  2.0 (4.0)  4.0 (64)  Amphotericin B  2.0 (8.0)  4.0 (16)  2.0 (4.0)  <1.0 (1.0)  8.0 (>64)  Caspofungin  2.0 (8.0)  4.0 (32)  8.0 (32)  8.0 (64)  8.0 (100)  Fluconazole  64 (200)  64 (>200)  64 (>200)  32 (200)  100 (>200)    SMIC50 (SMIC80)   Antimicrobial  C. albicans  C. auris  C. auris  C. auris  C. auris  ATCC 90028  CDC381  CDC383  CDC386  CDC390  CSA-44  2.0 (8.0)  4.0 (16)  2.0 (8.0)  2.0 (4.0)  4.0 (64)  CSA-131  2.0 (4.0)  2.0 (32)  2.0 (4.0)  2.0 (4.0)  4.0 (64)  Amphotericin B  2.0 (8.0)  4.0 (16)  2.0 (4.0)  <1.0 (1.0)  8.0 (>64)  Caspofungin  2.0 (8.0)  4.0 (32)  8.0 (32)  8.0 (64)  8.0 (100)  Fluconazole  64 (200)  64 (>200)  64 (>200)  32 (200)  100 (>200)  Table 3. Susceptibility profiles (mg/L) of sessile fungi (biofilm) to CSA-44, CSA-131 and three antifungal compounds   SMIC50 (SMIC80)   Antimicrobial  C. albicans  C. auris  C. auris  C. auris  C. auris  ATCC 90028  CDC381  CDC383  CDC386  CDC390  CSA-44  2.0 (8.0)  4.0 (16)  2.0 (8.0)  2.0 (4.0)  4.0 (64)  CSA-131  2.0 (4.0)  2.0 (32)  2.0 (4.0)  2.0 (4.0)  4.0 (64)  Amphotericin B  2.0 (8.0)  4.0 (16)  2.0 (4.0)  <1.0 (1.0)  8.0 (>64)  Caspofungin  2.0 (8.0)  4.0 (32)  8.0 (32)  8.0 (64)  8.0 (100)  Fluconazole  64 (200)  64 (>200)  64 (>200)  32 (200)  100 (>200)    SMIC50 (SMIC80)   Antimicrobial  C. albicans  C. auris  C. auris  C. auris  C. auris  ATCC 90028  CDC381  CDC383  CDC386  CDC390  CSA-44  2.0 (8.0)  4.0 (16)  2.0 (8.0)  2.0 (4.0)  4.0 (64)  CSA-131  2.0 (4.0)  2.0 (32)  2.0 (4.0)  2.0 (4.0)  4.0 (64)  Amphotericin B  2.0 (8.0)  4.0 (16)  2.0 (4.0)  <1.0 (1.0)  8.0 (>64)  Caspofungin  2.0 (8.0)  4.0 (32)  8.0 (32)  8.0 (64)  8.0 (100)  Fluconazole  64 (200)  64 (>200)  64 (>200)  32 (200)  100 (>200)  Confocal laser scanning microscopy of fungal biofilms To observe the antibiofilm properties of a lead ceragenin, biofilms of C. albicans and C. auris were treated with CSA-131, stained and imaged via confocal microscopy (Figure 3). Untreated biofilms exhibited expected aggregates of live cells, while treated biofilms showed comparable aggregates of dead cells. As with Gram-negative bacteria,49 the ceragenin was able to penetrate the extracellular matrix and exert antifungal activity without significantly compromising the biofilm morphology. The antibiofilm property of the ceragenins complements their ability to inhibit biofilm formation as reported in a recent study in which CSA-131, incorporated into a hydrogel, prevents biofilm formation on a medical device for extended periods.50 Figure 3. View largeDownload slide Confocal laser scanning micrographs (×60 magnification) of stained fungal biofilms. Green: live cells; red: dead cells. (a) C. albicans, untreated. (b) C. albicans, treated with CSA-131 (50 mg/L). (c) C. auris, untreated. (d) C. auris, treated with CSA-131 (50 mg/L). Figure 3. View largeDownload slide Confocal laser scanning micrographs (×60 magnification) of stained fungal biofilms. Green: live cells; red: dead cells. (a) C. albicans, untreated. (b) C. albicans, treated with CSA-131 (50 mg/L). (c) C. auris, untreated. (d) C. auris, treated with CSA-131 (50 mg/L). Antifungal activity of formulated ceragenins in tissue explants There are many potential applications of novel antifungal agents and some of the most widely useful would involve topical application to skin or mucosal tissues. For these types of applications, lead ceragenins would need to be formulated into gels or creams. HEC (typically used at 2.7% in water) can be used to form a lubricating gel into which antimicrobial compounds can be formulated. Pluronic® F-127 is a non-ionic surfactant that has shown compatibility with ceragenins with concomitant decreases in cytotoxic properties.51,52 Combining HEC, Pluronic® F-127 and either CSA-44 or CSA-131 at 0.5% (CSA-44L and CSA-131L) or 2.0% (CSA-44H and CSA-131H) gave stable, lubricious gels. These percentages of ceragenins are comparable to those of antifungals incorporated into commercially available antifungal products. Porcine vaginal mucosal explants were used to determine the antifungal activities of these gels. Nystatin (a polyene antifungal related to amphotericin B) was used as a comparator and the HEC/Pluronic® F-127 combination (vehicle) was used to observe effects of these compounds on the fungi. Fungal infection with either C. albicans or C. auris was established over 2 and 24 h of incubation, followed by treatment for 24 h with the test formulations. Over this time course, fungal populations grew to over 106 cfu/explant (Figure 4). In all experiments, the vehicle had no impact on fungal growth. Figure 4. View largeDownload slide Antifungal activities of nystatin (100 000 USP nystatin units) compared with HEC/Pluronic® F-127 formulations of CSA-44 and CSA-131 (CSA-44L and CSA-131L contain 0.5% ceragenin and CSA-44H and CSA-131H contain 2% ceragenin) in porcine vaginal mucosal tissue explants. Log10 cfu reduction from untreated growth control following 2 h (a and b) and 24 h (c and d) infections of C. auris or C. albicans on PVM. Data presented are means and SEMs. Analyses of variance (ANOVA) followed by Dunnett’s multiple comparison post-test were performed using the GraphPad PRISM software. *Significantly different from growth control (P < 0.5); #Significantly different from nystatin (P < 0.5). Figure 4. View largeDownload slide Antifungal activities of nystatin (100 000 USP nystatin units) compared with HEC/Pluronic® F-127 formulations of CSA-44 and CSA-131 (CSA-44L and CSA-131L contain 0.5% ceragenin and CSA-44H and CSA-131H contain 2% ceragenin) in porcine vaginal mucosal tissue explants. Log10 cfu reduction from untreated growth control following 2 h (a and b) and 24 h (c and d) infections of C. auris or C. albicans on PVM. Data presented are means and SEMs. Analyses of variance (ANOVA) followed by Dunnett’s multiple comparison post-test were performed using the GraphPad PRISM software. *Significantly different from growth control (P < 0.5); #Significantly different from nystatin (P < 0.5). At 2 h, nystatin decreased fungal counts with C. auris and C. albicans by ∼1 log, but only with C. albicans was this reduction statistically significant. In contrast, both ceragenins, at both concentrations, significantly reduced fungal counts at 2 h, with reductions as great as 5 logs (CSA-131H). At 24 h, nystatin-treated explants supported fungal counts comparable to controls with both species of fungi. At 24 h, the most potent activity of ceragenins was observed with C. albicans, with fungal counts significantly reduced relative to both control and nystatin-treated explants. With the highest concentrations of ceragenin (2%), fungal counts were reduced by >4 logs. Although the activity of the ceragenins at 24 h was not as dramatic with explants colonized with C. auris, significant reductions (∼3 logs) were observed with the highest concentration of the ceragenins. Significant reduction was also observed with the lower concentration (0.5%) of CSA-44. CSA-44 was also formulated into a cream and compared with a commercial minocycline product, Monistat 7, in the porcine vaginal mucosa (PVM) infection model. Based on efficacy of the ceragenins in the gel formulation, a concentration of 1% CSA-44 in a cream was used and compared with Monistat 7 (2% miconazole in cream formulation). No other formulations were evaluated in this assay. When tested for antifungal activity, the cream vehicle did not impact growth (i.e. growth of C. albicans in the presence of the cream formulation was not different from a blank control.) With C. albicans, Monistat 7 reduced fungal counts by a statistically significant amount at 2 h, compared with the growth control, but this effect was negligible at 24 h. With this organism, the CSA-44 cream formulation reduced counts by >3 logs at 2 h and by ∼2 logs at 24 h; both of these measurements were significantly different from both the vehicle and the Monistat 7-treated explants (Figure 5). Explants colonized with C. auris were not significantly affected by Monostat-7 treatment at 2 and 24 h. In contrast, the CSA-44-containing cream significantly reduced C. auris counts at both timepoints (>2 logs and >1 log reductions, respectively). Figure 5. View largeDownload slide Antifungal activities of Monistat 7 (2% miconazole) compared with a cream formulation of CSA-44 (1%) in porcine vaginal mucosal tissue explants. Log10 cfu reduction from untreated growth control following 2 h (a and b) and 24 h (c and d) infections of C. auris or C. albicans on PVM. Data presented are means and SEMs. Analyses of variance (ANOVA) followed by Dunnett’s multiple comparison post-test were performed using the GraphPad PRISM software. *Significantly different from growth control (P < 0.5); ^Significantly different from Monistat 7 (P < 0.5). Figure 5. View largeDownload slide Antifungal activities of Monistat 7 (2% miconazole) compared with a cream formulation of CSA-44 (1%) in porcine vaginal mucosal tissue explants. Log10 cfu reduction from untreated growth control following 2 h (a and b) and 24 h (c and d) infections of C. auris or C. albicans on PVM. Data presented are means and SEMs. Analyses of variance (ANOVA) followed by Dunnett’s multiple comparison post-test were performed using the GraphPad PRISM software. *Significantly different from growth control (P < 0.5); ^Significantly different from Monistat 7 (P < 0.5). Conclusions The spread of C. auris, the high mortality rates in patients with infection and the high prevalence of drug resistance among clinical isolates underscore the need for the development of novel antifungal agents. The mechanisms common to endogenous AMPs provide direction for investigation of therapeutic agents that mimic antimicrobial activities that nature has honed over millions of years. Due to their simple structures, ceragenins are attractive candidates for development of novel antifungal agents; they can be prepared on a large scale and they are stable in the presence of ubiquitous proteases. The ceragenins display antifungal activities with C. auris comparable to the most active agents tested and they do not show cross-resistance with other antifungals. The ceragenins also retain activity against sessile organisms in established biofilms. In formulated forms, ceragenins are active against high inocula of fungi in mucosal tissue. The substantial antifungal activities of these ceragenin formulations compared with nystatin and Monistat 7 highlight their potential for use as novel antifungals for topical or mucosal applications. Acknowledgements We thank N8 Medical, Inc., CSA Biotechnologies and Brigham Young University for generous funding. Funding This study was supported by funding from N8 Medical, Inc., CSA Biotechnologies and Brigham Young University. Transparency declarations P. B. S. is a paid consultant for N8 Medical, Inc. All other authors: none to declare. References 1 Lockhart SR, Etienne KA, Vallabhanemi S et al.   Simultaneous emergence of multidrug-resistant Candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses. Clin Infect Dis  2017; 64: 134– 40. Google Scholar CrossRef Search ADS PubMed  2 Clancy CJ, Nguyen MH. Emergence of Candida auris: an international call to arms. Clin Infect Dis  2017; 64: 141– 3. Google Scholar CrossRef Search ADS PubMed  3 Chowdhary A, Sharma C, Meis JF. Candida auris: a rapidly emerging cause of hospital-acquired multidrug-resistant fungal infections globally. PLoS Pathog  2017; 13: e1006290. Google Scholar CrossRef Search ADS PubMed  4 Todd B. Clinical alert: Candida auris. Am J Nurs  2017; 117: 53– 5. Google Scholar CrossRef Search ADS PubMed  5 Sarma S, Upadhyay S. Current perspective on emergence, diagnosis and drug resistance in Candida auris. Infect Drug Resist  2017; 10: 155– 65. Google Scholar CrossRef Search ADS PubMed  6 Tsay S, Welsh RM, Adams EH et al.   Ongoing transmission of Candida auris in health care facilities — United States, June 2016–May 2017. MMWR Morb Mortal Wkly Rep  2017; 66: 514– 5. Google Scholar CrossRef Search ADS PubMed  7 Mohsin J, Hagen F, Al-Balushi ZAM et al.   The first cases of Candida auris candidaemia in Oman. Mycoses  2017; 60: 569– 75. Google Scholar CrossRef Search ADS PubMed  8 Rudramurthy SM, Chakrabarti A, Paul RA et al.   Candida auris candidaemia in Indian ICUs: analysis of risk factors. J Antimicrob Chemother  2017; 72: 1794– 801. Google Scholar CrossRef Search ADS PubMed  9 Arendrup MC, Prakash A, Meletiadis J et al.   Comparison of EUCAST and CLSI reference microdilution MICs of eight antifungal compounds for Candida auris and associated epidemiological cutoff values. Antimicrob Agents Chemother  2017; 61: e00485– 17. Google Scholar PubMed  10 McCarthy MW, Walsh TJ. Drug development challenges and strategies to address emerging and resistant fungal pathogens. Exp Rev Anti-Infect Therapy  2017; 15: 577– 84. Google Scholar CrossRef Search ADS   11 Silva PM, Gonçalves S, Santos NC. Defensins: antifungal lessons from eukaryotes. Front Microbiol  2014; 5: 97. Google Scholar PubMed  12 Swidergall M, Ernst JF. Interplay between Candida albicans and the antimicrobial peptide armory. Eukaryot Cell  2014; 13: 950– 7. Google Scholar CrossRef Search ADS PubMed  13 Duncan VMS, O’Neil DA. Commercialization of antifungal peptides. Fungal Biol Rev  2013; 26: 156– 65. Google Scholar CrossRef Search ADS   14 Vriens K, Cammue BPA, Thevissen K. Antifungal plant defensins: mechanisms of action and production. Molecules  2014; 19: 12280– 303. Google Scholar CrossRef Search ADS PubMed  15 Hashemi MM, Holden BS, Durnas B et al.   Ceragenins as mimics of endogenous antimicrobial peptides. J Antimicrob Agents  2017; 3: 1000141. Google Scholar CrossRef Search ADS   16 Lai X-Z, Feng Y, Pollard J et al.   Ceragenins: cholic acid-based mimics of antimicrobial peptides. Acc Chem Res  2008; 41: 1233– 40. Google Scholar CrossRef Search ADS PubMed  17 Hashemi MM, Rovig J, Weber S et al.   Susceptibility of colistin-resistant, Gram-negative bacteria to antimicrobial peptides and ceragenins. Antimicrob Agents Chemother  2017; 61: e00292-17. Google Scholar CrossRef Search ADS PubMed  18 Vila-Farrés X, Callarisa AE, Gu X et al.   CSA-131, a ceragenin active against colistin-resistant Acinetobacter baumannii and Pseudomonas aeruginosa clinical isolates. Int J Antimicrob Agents  2015; 46: 568– 71. Google Scholar CrossRef Search ADS PubMed  19 Olekson MA, You T, Savage PB et al.   Antimicrobial ceragenins inhibit biofilms and affect mammalian cell viability and migration in vitro. FEBS Open Bio  2017; 7: 953– 67. Google Scholar CrossRef Search ADS PubMed  20 Durnas B, Wnorowska U, Pogoda K et al.   Candidacidal activity of selected ceragenins and human cathelicidin LL-37 in experimental settings mimicking infection sites. PLoS One  2016; 11: e0157242. Google Scholar CrossRef Search ADS PubMed  21 Kodedová M, Sychrová H. Synthetic antimicrobial peptides of the halcitines family disturb the membrane integrity of Candida cells. Biochim Biophys Acta  2017; 1859: 1851– 8. Google Scholar CrossRef Search ADS PubMed  22 Mangoni ML, Luca V, McDermott AM. Fighting microbial infections: a lesson from amphibian skin-derived esculentin-1 peptides. Peptides  2015; 71: 286– 95. Google Scholar CrossRef Search ADS PubMed  23 Tsai PW, Cheng YL, Hsieh WP et al.   Responses of Candida albicans to the human antimicrobial peptide LL-37. J Microbiol  2014; 52: 581– 9. Google Scholar CrossRef Search ADS PubMed  24 Lee H, Hwang JS, Lee DG. Scolopendin, an antimicrobial peptide from centipede, attenuates mitochondrial functions and triggers apoptosis in Candida albicans. Biochem J  2017; 474: 635– 45. Google Scholar CrossRef Search ADS PubMed  25 Lee J, Lee DG. Melittin triggers apoptosis in Candida albicans through the reactive oxygen species-mediated mitochondria/caspase-dependent pathway. FEMS Microbiol Lett  2014; 355: 36– 42. Google Scholar CrossRef Search ADS PubMed  26 Wong JH, Ng TB, Legowska A et al.   Antifungal action of human cathelicidin fragment (LL13-37) on Candida albicans. Peptides  2011; 32: 1996– 2002. Google Scholar CrossRef Search ADS PubMed  27 Park C, Lee DG. Melittin induces apoptotic features in Candida albicans. Biochem Biophys Res Commun  2010; 394: 170– 2. Google Scholar CrossRef Search ADS PubMed  28 Niemirowicz K, Durnaś B, Tokajuk G et al.   Formulation and candidacidal activity of magnetic nanoparticles coated with cathelicidin LL-37 and ceragenin CSA-13. Sci Rep  2017; 7: 4610. Google Scholar CrossRef Search ADS PubMed  29 Schindeler A, Yu NYC, Cheng TL et al.   Local delivery of the cationic steroid antibiotic CSA-90 enables osseous union in a rat open fracture model of Staphylococcus aureus infection. J Bone Joint Surg Am  2015; 97: 302– 9. Google Scholar CrossRef Search ADS PubMed  30 Bucki R, Niemirowicz K, Wnorowska U et al.   Bactericidal activity of ceragenin CSA-13 in cell culture and in an animal model of peritoneal infection. Antimicrob Agents Chemother  2015; 59: 6274– 82. Google Scholar CrossRef Search ADS PubMed  31 Sinclair CD, Pham TX, Williams DL et al.   Model development for determining the efficacy of a combination coating for the prevention of perioperative device related infections: a pilot study. J Biomed Mater Res B Appl Biomater  2013; 101: 1143– 53. Google Scholar CrossRef Search ADS PubMed  32 Williams DL, Haymond BS, Beck PJ et al.   In vivo efficacy of a silicone-cationic steroid antimicrobial coating to prevent implant-related infection. Biomaterials  2012; 33: 8641– 56. Google Scholar CrossRef Search ADS PubMed  33 Ding B, Yin N, Liu Y et al.   Origins of cell selectivity of cationic steroid antibiotics. J Am Chem Soc  2004; 126: 13642– 8. Google Scholar CrossRef Search ADS PubMed  34 Guan Q, Schmidt EJ, Boswell SR et al.   Preparation and characterization of cholic acid-derived antimicrobial agents with controlled stabilities. Org Lett  2000; 2: 2837– 40. Google Scholar CrossRef Search ADS PubMed  35 Boikov DA, Locke JB, James KD et al.   In vitro activity of the novel echinocandin CD101 at pH 7 and 4 against Candida spp. isolates from patients with vulvovaginal candidiasis. J Antimicrob Chemother  2017; 72: 1355– 8. Google Scholar CrossRef Search ADS PubMed  36 De Seta F, Schmidt M, Vu B et al.   Antifungal mechanisms supporting boric acid therapy of Candida vaginitis. J Antimicrob Chemother  2009; 63: 325– 36. Google Scholar CrossRef Search ADS PubMed  37 Valentín A, Cantón E, Pemán J et al.   In vitro activity of anidulafungin in combination with amphotericin B or voriconazole against biofilms of five Candida species. J Antimicrob Chemother  2016; 71: 3449– 52. Google Scholar CrossRef Search ADS PubMed  38 Zhang Y, Chen Y-Y, Huang L et al.   The antifungal effects and mechanical properties of silver bromide/cationic polymer nano-composite-modified Poly-methyl methacrylate-based dental resin. Sci Rep  2017; 7: 1547. Google Scholar CrossRef Search ADS PubMed  39 Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Third Informational Supplement M27-A3 . CLSI, Wayne, PA, USA, 2008. 40 Sóczó G, Kardos G, McNicholas PM et al.   Correlation of posaconazole minimum fungicidal concentration and time–kill test against nine Candida species. J Antimicrob Chemother  2007; 60: 1004– 9. Google Scholar CrossRef Search ADS PubMed  41 Moss BJ, Kim Y, Nandakumar MP et al.   Quantifying metabolic activity of filamentous fungi using a colorimetric XTT assay. Biotechnol Prog  2008; 24: 780– 3. Google Scholar CrossRef Search ADS PubMed  42 Anderson MJ, Parks PJ, Peterson ML. A mucosal model to study microbial biofilm development and anti-biofilm therapeutics. J Microbiol Methods  2013; 92: 201– 8. Google Scholar CrossRef Search ADS PubMed  43 Bruzual I, Riggle P, Hadley S et al.   Biofilm formation by fluconazole-resistant Candida albicans strains is inhibited by fluconazole. J Antimicrob Chemother  2007; 59: 441– 50. Google Scholar CrossRef Search ADS PubMed  44 Kagan S, Jabbour A, Sionov E et al.   Anti-Candida albicans biofilm effect of novel heterocyclic compounds. J Antimicrob Chemother  2014; 69: 416– 27. Google Scholar CrossRef Search ADS PubMed  45 Khan MSA, Ahmad I. Antibiofilm activity of certain phytocompounds and their synergy with fluconazole against Candida albicans biofilms. J Antimicrob Chemother  2012; 67: 618– 21. Google Scholar CrossRef Search ADS PubMed  46 Lazzell AL, Chaturvedi AK, Pierce CG et al.   Treatment and prevention of Candida albicans biofilms with caspofungin in a novel central venous catheter murine model of candidiasis. J Antimicrob Chemother  2009; 64: 567– 70. Google Scholar CrossRef Search ADS PubMed  47 Larkin E, Hager C, Chandra J et al.   The emerging Candida auris: characterization of growth phenotype, virulence factors, antifungal activity, and effect of SCY-078, a novel glucan synthesis inhibitor, on growth morphology and biofilm formation. Antimicrob Agents Chemother  2017; 61: e02396– 16. Google Scholar CrossRef Search ADS PubMed  48 Oh BJ, Shin JH, Kim M-N et al.   Biofilm formation and genotyping of Candida haemulonii, Candida pseudohaemulonii, and a proposed new species (Candida auris) isolates from Korea. Med Mycol  2011; 49: 98– 102. Google Scholar CrossRef Search ADS PubMed  49 Nagant C, Pitts B, Stewart PS et al.   Study of the effect of antimicrobial peptide mimic, CSA-13, on an established biofilm formed by Pseudomonas aeruginosa. Microbiol Open  2013; 2: 318– 25. Google Scholar CrossRef Search ADS   50 Hashemi MM, Rovig J, Bateman J et al.   Preclinical testing of a broad-spectrum antimicrobial endotracheal tube coated with an innate immune synthetic mimic. J Antimicrob Chemother  2018; 73: 143– 50. Google Scholar CrossRef Search ADS PubMed  51 Nagant C, Savage PB, Dehaye JP. Effect of pluronic acid F-127 on the toxicity towards eukaryotic cells of CSA-13, a cationic steroid analogue of antimicrobial peptides. J Appl Microbiol  2012; 112: 1173– 83. Google Scholar CrossRef Search ADS PubMed  52 Leszczyńska K, Namiot A, Cruz K et al.   Potential of ceragenin CSA-13 and its mixture with pluronic F-127 as treatment of topical bacterial infections. J Appl Microbiol  2011; 110: 229– 38. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Apr 4, 2018

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