The Emerging Pathogen Candida auris: Growth Phenotype, Virulence Factors, Activity of Antifungals, and Effect of SCY-078, a Novel Glucan Synthesis Inhibitor, on Growth Morphology and Biofilm Formation

Isolates tested.The following C. auris strains were used in this study: 2 C. auris isolates (MRL 31102 and MRL 31103) obtained from a 68-year-old male enrolled in a recent candidemia trial at a German site and 14 C. auris isolates from the CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands (CBS 10913, CBS 12372, CBS 12373, CBS 12766 to CBS 12768, and CBS 12770 to CBS 12777) that were isolated from patients living in Japan, India, and South Korea (Table 4). To identify the MRL 31102 and MRL 31103 isolates, we originally utilized the yeast identification tool API 20C AUX. However, we were unable to identify the strains with a high percentage (>85%) of certainty. Moreover, since C. auris is also often misidentified as Candida haemulonii, Candida famata, Candida sake, or Rhodotorula glutinis using typical identification methods, including the API 20C AUX tool and the Vitek 2 system, we used DNA typing, employing the internal transcribed spacer 1 (ITS1) and ITS4 regions of the fungal ribosome (2–6, 12–15). Identification was performed using a BLAST algorithm search against the sequences in the UNITE database (32). This analysis allowed us to identify MRL 31102 and MRL 31103, which had 89 and 99% homology with the C. auris strains in the UNITE database, respectively, as C. auris. In the current study, two strains of C. albicans (SC5314 and ATCC 14053), obtained from the American Type Culture Collection (Manassas, VA), were used as comparators. C. albicans was selected as a comparator because its virulence factors have been well characterized and it is responsible for nearly 50% of all Candida infections.

Ability of C. auris to form chlamydospores.Since chlamydospores are utilized to help distinguish fungal types (33), we evaluated the ability of the C. auris isolates to exhibit this morphotype. Briefly, an isolated colony of C. auris was inoculated onto a cornmeal agar plate (Becton Dickinson, Sparks, MD, USA) to make a lawn. A coverslip was applied to the inoculated area in order to create microaerophilic conditions, and the plate was incubated at 30°C for 3 days with protection from light. The plate was subsequently examined for chlamydospore formation under white light at a ×20 magnification. C. albicans ATCC 14053 was used as a positive control.

Characterization of C. auris virulence factors.The ability of Candida species to cause infection is attributed to the possession of virulence factors, including the ability to germinate, adhere to host cells, secrete extracellular enzymes such as phospholipase and proteinase, and form biofilms (18, 34, 35). To date, the characterization of C. auris virulence factors is limited.

(i) Ability to germinate.The ability of C. auris to form germ tubes was assessed using the germ tube test (36–38). Briefly, an isolated colony of C. auris, taken from a 24-h culture grown on potato-dextrose agar (Becton Dickinson, Sparks, MD, USA), was inoculated into 0.5 ml of fetal bovine serum (Fisher Thermo Scientific, Cleveland, OH, USA) and incubated for 2 h at 37°C. Next, 10 μl of fetal bovine serum was then transferred onto a glass slide, a coverslip was added, and the slide was examined using phase-contrast microscopy at a ×20 magnification.

(ii) Adherence assay.The ability of Candida species to adhere to indwelling medical devices and host tissues is a known virulence factor. Since silicon elastomer is used extensively in central venous catheters, in this study, we evaluated the ability of C. auris isolates to adhere to silicon elastomer, which was used as a representative catheter material.

To determine the ability of C. auris to adhere to silicon elastomer surfaces, yeast cells were inoculated into 10 ml yeast nitrogen base broth (Becton Dickinson, Sparks, MD, USA) and incubated overnight at 37°C. The cells were then washed twice with Hanks' buffered salt solution, adjusted to 5 × 10³ cells/ml, and further diluted to obtain a cell suspension of 250 cells/ml using the same buffer. Silicon elastomer discs (diameter, 1.2 cm) were transferred to 12-well tissue culture plates, to which 2 ml of cell suspension (prepared as described above) was added. Next, the plates were incubated at 37°C for 30 min to allow the cells to adhere to the discs. After incubation, discs containing adherent cells were transferred to a fresh plate containing 2 ml Hanks' buffered salt solution. Subsequently, the discs were washed twice with Hanks' buffered salt solution and, following aspiration of the buffer, were overlaid with 3 ml of Sabouraud dextrose agar (Becton Dickinson, Sparks, MD, USA). The plates were incubated at 37°C for 18 to 24 h, and the number of C. auris cells adhering to the catheter material was assessed by counting the number of CFU.

(iii) Phospholipase assay.The ability of the C. auris strains to secrete phospholipase was determined using a phospholipase activity assay that employed Sabouraud dextrose agar as a medium supplemented with 58.4 g/liter NaCl, 5.5 g/liter CaCl₂, and 10% egg yolk emulsion (30% stock; Hi-Media, India) as previously described (26, 39). Medium without egg yolk was sterilized at 121°C for 15 min and cooled to 50°C. Egg yolk emulsion was then added, and the agar was dispensed into petri dishes (20 ml/plate). A 10-μl aliquot containing 1 × 10⁷ yeast cells/ml was added to the center of the plates, and the plates were incubated at 37°C for 5 days. Following incubation, the production of phospholipase (indicated by the appearance of a whitish zone of precipitation around the yeast colony) was assessed by measuring the ratio of the colony diameter to the colony diameter plus the precipitation zone (the P_z value). P_z values of ≤0.89, 0.90 to 0.99, and 1 indicate strong, weak, and no phospholipase activity, respectively (26).

(iv) Proteinase assay.Proteinase activity assays were performed using a protease fluorescent detection kit (catalog number PF0100; Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's specifications (40). The C. auris strains were cultured in 20 ml yeast nitrogen base for 48 h. Following incubation, the supernatant containing secreted proteinases was concentrated, and the volumes were adjusted to the proportion of the total number of CFU. Subsequently, 10 μl of the normalized protein-rich supernatants was tested for proteinase activity using the kit described above.

(v) Biofilm formation.The ability of Candida to form biofilms has been linked to catheter-associated infections. Therefore, we evaluated whether C. auris can produce biofilms using our in vitro biofilm model as described previously (19). In the current study, we used silicone elastomer as a substrate for biofilm formation since catheters are commonly constructed using this material and silicone elastomer has been used as a substrate in numerous studies investigating catheter-associated biofilms performed by our group and others (41–51). Briefly, silicone elastomer discs (diameter, 1.2 cm) were placed in 12-well tissue culture plates and incubated in fetal bovine serum at 37°C on a rocker for 24 h. The discs were then removed from the fetal bovine serum, immersed in a 4-ml cell suspension with a concentration of 1 × 10⁷ cells/ml, and incubated for 90 min at 37°C (adhesion phase). Subsequently, the discs were transferred to 4 ml yeast nitrogen base medium and incubated for 24 h at 37°C to form mature biofilms (mature phase) (19).

The quantification of the biofilms formed by different strains was performed using a colorimetric metabolic assay (to measure mitochondrial dehydrogenase activity) and dry weight analysis (to measure total biofilm mass, which includes fungal cells and matrix). For evaluation of metabolic activity, discs with biofilms were transferred to 12-well tissue culture plates containing 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT; 12.5 μg/ml) and menadione (1 μM) in phosphate-buffered saline and incubated at 37°C for 3 h as described previously (19). Next, the biofilms were scraped, transferred into a tube, and centrifuged at 3,000 rpm for 15 min. The absorbance of the resulting supernatant was measured spectrophotometrically at 492 nm. To determine dry weight (biomass), the pellet obtained from the centrifugation step described above was resuspended in phosphate-buffered saline and filtered through a preweighed filter (pore size, 0.2 μm; Millipore, Billerica, MA, USA), dried at 37°C for 48 h, and weighed (19). The biomass of the biofilms was calculated from the difference in the weight of the preweighed filters.

To examine the morphology and thickness of the formed biofilms, confocal scanning laser microscopy was used. Briefly, following biofilm formation, the silicon elastomer discs on which the biofilms were developed were transferred to a 35-mm-diameter glass-bottom petri dish (MatTek Corp., Ashland, MA, USA) and incubated for 45 min at 37°C in 2 ml of phosphate-buffered saline containing the fluorescent stains FUN-1 (10 μM) and concanavalin A (ConA; 25 mg/liter)-Alexa Fluor 488 conjugate (both dyes from Molecular Probes, Inc., Eugene, OR). FUN-1 (excitation wavelength, 543 nm; emission wavelength, 560 nm) is converted to orange-red cylindrical intravacuolar structures by metabolically active cells, and ConA (excitation wavelength, 488 nm; emission wavelength, 505 nm) fluoresces green when bound to glucose and mannose residues of fungal polysaccharides (present in the cell wall and biofilm matrix). After incubation with the dyes, the silicon elastomer discs were flipped and the biofilms were examined using a Zeiss LSM510 confocal scanning laser microscope (Carl Zeiss, Inc.). To determine the structure of the biofilms, a series of horizontal (x-y) optical sections with a thickness of 0.9 μm were taken at 0.44-μm intervals throughout the full length of the biofilm. Confocal images of green (ConA) and red (FUN-1) fluorescence were simultaneously collected using a multitrack mode (19).

Determination of susceptibility profiles of C. auris strains.To establish the susceptibility profiles of the C. auris strains, broth microdilution susceptibility testing was performed in accordance with the guidelines in the Clinical and Laboratory Standards Institute (CLSI) M27-A3 document (52). RPMI 1640 medium with 3-(N-morpholino)propanesulfonic acid (RPMI), an inoculum of 0.5 × 10³ to 2.5 × 10³ cells/ml, and incubation at 35°C were used. The activities of 11 antifungals against the C. auris isolates were tested, including SCY-078 (Scynexis, Jersey City, NJ, USA), a novel orally bioavailable 1,3-β-d-glucan synthesis inhibitor, and currently available antifungals amphotericin B and anidulafungin (Pfizer Pharmaceuticals, New York, NY, USA), caspofungin (Merck Co., Kenilworth, NJ, USA), fluconazole, flucytosine, and isavuconazole (Astellas Pharma US, Inc., Northbrook, IL, USA), itraconazole and micafungin (Astellas Pharma, Inc., Tokyo, Japan), posaconazole, and voriconazole (AMB, FLC, 5FC, ITC, POS, and VRC were obtained from Sigma-Aldrich, St. Louis, MO, USA). MIC panels were incubated for 24 h (SCY-078, AMB, AFG, CAS, FLC, ISA, MFG, and VRC) or 48 h (5FC, AMB, FLC, ITC, POS, and VRC). MIC endpoints were determined visually as the lowest concentration of drug that resulted in the complete inhibition of growth (AMB) or a decrease of growth by ≥50% relative to that of the growth control (SCY-078, AFG, CAS, FLC, 5FC, ISA, ITC, MFG, POS, VRC) (52). In all instances, MIC plates were prepared using reagent-grade powders, as directed by CLSI (52).

Effect of SCY-078 on the growth of C. auris and C. albicans.Evaluation of the ability of a drug to inhibit microbial growth is conventionally determined either by counting the number of CFU, which is an indicator of cell viability, or by turbidity measurement (using a spectrophotometer), which indicates cell density but does not differentiate between live and dead cells (52, 53). In this study, we used the spectrophotometric approach to evaluate the ability of SCY-078 to inhibit Candida growth as described previously (54). Briefly, cells were harvested from 18- to 24-h-old cultures, washed twice with phosphate-buffered saline, and adjusted to 5 × 10⁵ cells/ml in 50-ml conical tubes containing RPMI alone (with no drug as a growth control) or 0.5×, 1×, 2×, 4×, 8×, or 16× MIC of SCY-078. Medium alone with no drug or yeast cells was used as a blank. All tubes were incubated at 37°C. At different time points (0, 5, 8, 24, 30, 48, and 54 h), aliquots were taken and their optical densities (ODs) were determined spectrophotometrically at 520 nm. A growth curve showing cell inhibition temporally was constructed.

Scanning electron microscopy.The effect of SCY-078 on C. auris morphology and ultrastructure was determined using scanning electron microscopy as described previously (19). Briefly, the C. auris strains were exposed overnight to 1× MIC (0.5 mg/liter) of SCY-078 at 35°C. Next, 200 μl of a cell suspension was fixed in 2% glutaraldehyde, and the fixed cell suspension was incubated at 4°C for 48 h. After fixation, the samples were processed and dried. The processed samples were coated with palladium for 60 s and viewed with a Nova NanoLab 200 FEG-SEM/FIB scanning electron microscope in high-vacuum mode at 2.00 kV.

Effect of SCY-078 on C. auris biofilms.To evaluate the activity of SCY-078 against C. auris biofilms, discs with mature biofilms were transferred to wells containing different concentrations of SCY-078 (range, 0.5 to 4 mg/liter). Following 48 h of incubation, the metabolic activities of the biofilms were measured using the XTT reduction assay as described above. Images and the thicknesses of biofilms growing in the presence or absence of drug were captured using confocal scanning laser microscopy, also as described above (19).

Statistical analysis.Statistical analyses of all the data were performed using GraphPad Prism (version 6) software. The treated groups were compared to the untreated control groups using unpaired t tests, and a P value of <0.05 was considered statistically significant. All experiments were done in triplicate. Doubling times were calculated using R, the statistical programming language (https://cran.r-project.org/).

Accession number(s).The sequences of C. auris strains MRL 31102 and MRL 31103 have been deposited in GenBank under accession numbers KY514262 and KY514058, respectively.