Shining light on multi‐drug resistant Candida auris: Ultraviolet‐C disinfection, wavelength sensitivity, and prevention of biofilm formation of an emerging yeast pathogen

Abstract Candida auris is an emerging fungal superbug of worldwide interest. It is associated with high mortality rates and exhibits increased resistance to antifungals. Ultraviolet subtype C (UVC) light can be used to disinfect surfaces to mitigate its spread. The objectives of this study were (1) To investigate UVC disinfection performances and wavelength sensitivity of C. auris. (2) To evaluate the UVC dose required for the prevention of biofilm formation on stainless‐steel, plastic (polystyrene), and poly‐cotton fabric surfaces. C. auris was grown following standard procedures. The study utilized six different UVC LED arrays with wavelengths between 252 and 280 nm. Arrays were set at similar intensities, to obtain doses of 5–40 mJ cm−2 and similar irradiation time. Disinfection performance for each array was determined using log reduction value (LRV) and percentage reduction by comparing the controls against the irradiated treatments. Evaluation of the ability of 267 nm UVC LEDs to prevent C. auris biofilm formation was investigated using stainless‐steel, plastic coupons, and poly‐cotton fabric. Peak sensitivity to UVC disinfection was between 267 and 270 nm. With 20 mJ cm−2, the study obtained ≥LRV3. On stainless‐steel coupons, 30 mJ cm−2 was sufficient to prevent biofilm formation, while on plastic, this required 10 mJ cm−2. A dose of 60 mJ cm−2 reduced biofilms on poly‐cotton fabric significantly (R 2 = 0.9750, p = 0.0002). The study may allow for the design and implementation of disinfection systems.

It is currently a global emerging menace (Kordalewska & Perlin, 2019) that is considered to be an urgent threat by the Centers for Disease Control and Prevention (CDC) (Kadri, 2020) (Lone & Ahmad, 2019). Outbreaks have been documented in Europe (Ruiz-Gaitán et al., 2018), Asia , South America (Nobrega de Almeida et al., 2021), and North America (Prestel et al., 2021). The CDC reports an in-hospital mortality rate of up to 40% (Lemons et al., 2019), and the University of Maryland reports a mortality rate of 68% (Vila et al., 2020). Because of the high mortality rates, there is a need to not only have surveillance systems, such as those in New York (Zhu et al., 2020) but to also implement global mitigation strategies to help control the spread of C. auris (Ledwoch & Maillard, 2019). That is important because, unlike other Candida species, the deadly C. auris, which emerged simultaneously in three continents has had the unique ability that enabled it to spread globally, causing severe infections (Casadevall et al., 2019;Ku et al., 2018).
Of great consequence is that the emergence of C. auris may be a result of accelerating environmental and health trends (Chakrabarti & Sood, 2021). Although there has not been clear evidence that Covid-19 patients are more susceptible to C. auris infections, higher rates in Covid-19 treatment units such as the 39 cases have been reported in Florida by CDC during the pandemic (Prestel et al., 2021). Furthermore, the CDC has reported more outbreaks in Washington DC and Texas where 101 and 22 cases respectively were reported between January and April of 2021 . The emergence and spread of C. auris have a possible association with climate change (Casadevall et al., 2019). If this is the case, developing mitigation strategies immediately may be necessary to prevent wide-scale community outbreaks.
Of more concern is that C. auris displays several features that make mitigation and disinfection difficult. Some of these features include the ability to spread rapidly, persistence in the colonization of the skin and high-touch surfaces (Horton Mark et al., 2020), and resistance to common disinfectants (Vila et al., 2020). C. auris is also resistant to conventional antibiotics such as fluconazole (Du et al., 2020;Zhu et al., 2020), and can survive for at least 28 days in the environment, all of which make it problematic in healthcare settings (Maslo et al., 2019). C. auris also causes bloodstream infections (Ruiz-Gaitán et al., 2018). Its biofilms likely contribute to pathogenicity and aid in the spreading in healthcare settings due to the formation of surface-adherent communities that can withstand desiccation and are resistant to antifungals .
Ultraviolet-C (UVC) light has been demonstrated as effective by numerous studies (Cadnum et al., 2018;Chatterjee et al., 2020;Fu et al., 2020;Lemons et al., 2019;Maslo et al., 2019;Ponnachan et al., 2019). However, no UVC dose, wavelength, biofilm, or environmental conditions are often reported so it is not possible to compare results between studies or to use results to design or administer disinfection systems. Unfortunately, also because methylation and other produced chemicals can protect DNA from dimerization, it is difficult to determine the sensitivity of C. auris from theoretical models. Thus, the objectives of this study were to determine the wavelength sensitivity of this emerging pathogen, and the performance of UVC on preventing the formation of biofilms on stainless-steel, plastic, and poly-cotton fabric to allow for the design and implementation of disinfection systems for these high-touch surfaces. to 265 nm, with around 5% power variation between test wavelengths (Gigahertz, 2021).
The disinfection stage was set up using a ring stand that was placed above the disinfection zone. Inoculum for C. auris was prepared by growing the strain in YM broth at 37°C for 24 h. This was followed by centrifugation and resuspension of yeast cells in fresh 1× phosphate-buffered saline (PBS) buffer (Cat # BP3994) (Fu et al., 2020). To standardize the resuspended culture, OD 600 was adjusted using GENESYS 30 visible spectrophotometer (Thermo Scientific) to 0.15. This was to ensure the same concentration (8-9 × 10 5 CFU/ml) of C. auris ATCC MYA-5001 was obtained for each test. Additional confirmation to ensure approximately the same concentrations of test culture was performed by measuring the turbidity of the suspension to ensure an equivalent of 0.5 McFarland using a Sper Scientific 860040 Turbidity Meter (Sper Scientific) (Srivastava & Ahmad, 2020).
To investigate UVC yeasticidal activities for each array, 500 µl of C. auris with OD 600 of 0.15 in sterile 1× PBS buffer was placed at the center of empty Petri dish and irradiated while in liquid phase at varying durations to obtain target constant doses of UVC for each array (Figure 2b). Then 500 µl of C. auris test culture for both irradiated and unirradiated (controls) were recovered using a pipette and spread plated on YM agar, and incubated at 37°C for 48 h. As in a previous study , colonies were then counted for use in the calculation of logarithmic reduction values (LRV) and % reduction by comparing the controls (not irradiated with UVC) against the UVC irradiated treatments. Experiments were performed in triplicate.

| Evaluation of UVC for prevention of biofilm formation against C. auris
Fresh C. auris cultures were made from freezer stocks by inoculating the YM broth and growing aerobically overnight at 37°C in baffled flasks in a shaker running at 180 rpm for 24 h. Overnight cultures were centrifuged once at 4500 rpm for 10 min to remove media and suspended in 1× PBS buffer before adjusting optical density at 600 (OD 600 ) to 0.15. Stainless-steel, plastic (polystyrene), and poly-cotton fabric coupons were then inoculated with 200 μl of the suspension. To allow for adherence of C. auris to the coupon surfaces, the inoculated materials were incubated for 1.5 h at 37°C while shaking at 100 rpm (Khan et al., 2012). Following the adhesion phase, the coupons were washed twice using 300 µl of 1× PBS buffer to remove loosely attached cells. The 267 nm LED array was driven at 500 mA and 6.43 V with a UVC intensity of 1.00 mW cm −2 confirmed using the optometer. To determine UVC dose that offers significant or total prevention of biofilm formation, UVC was applied for 5, 10, 30, and 40 s, to yield UVC doses of 5, 10, 30, and 40 mJ cm −2 respectively on the contaminated surfaces of stainless-steel and plastic coupons. For polycotton fabric, 30, 40, and 60 mJ cm −2 were used as target doses.
After irradiation, a total of 100 µl freshly made 1% YM broth, was added to all treatments. Experiments were done in triplicates.
Coupons were incubated at 37°C for 24 h at 90% relative humidity (RH) (Sanchez et al., 2016). Incubator RH adjustments were achieved using 35.89 g of NaCl (Fisher Scientific S271-500) in 100 ml of deionized water in a beaker. Onset HOBO loggers were placed in the incubation chambers to monitor and confirm RH conditions during the incubation period.

| Biofilm analysis using crystal violet assay
After 24 h the biofilms were harvested for analysis following a crystal violet assay modified protocol from Sherry et al. (2017) and Gulati et al. (2018). Specifically, coupons were processed by first rinsing them three times using 300 μl 1× PBS buffer, then air drying for 45 min. They were then stained using 100 µl of 1% crystal violet (Signa cat# V5265-500) for another 45 min. This was followed by washing them four times with 300 μl sterile water to remove excess stain. Destaining was performed using 1000 μl 30% glacial acetic acid solution (Merck Millipore Cat #1.59166.0500). After destaining, 1000 μl of the 30% glacial acetic acid (destaining solution) was used for OD measurement at 595 nm.

| Biofilm analysis using CFU/ml assay
After 24 h of biofilm growth, the coupons were rinsed three times in 300 μl 1× PBS buffer to rid of any planktonic bacteria. This was followed by the addition of 1 ml 1× PBS buffer for sonication. The sonicator (Branson Model # CPX 2800) was first degassed for 5 min, before sonication for 20 min, followed by vortexing to mix. Recovery was done by growing on YM agar. Incubation done at 37°C at 90% humidity for 48 h and colony counts used in statistical analysis.

| Optical microscopy
The surface properties of stainless-steel, plastic, and poly-cotton fabric coupons were investigated using an Olympus (BX41M-LED) microscope. Exposure time was set at 35, 60, and 175 ms respectively.

| Statistical analysis
To trace the trend related to disinfection performance for each array, linear regression analysis (simple exponential model) was carried out.
Additionally, an unpaired t-test (two-tailed) was used to measure the statistical significance of the impact of UVC in the prevention of F I G U R E 2 (a) Spectral analysis of arrays used in the study and (b) stage setup during the test biofilm formation. Statistical analysis was performed using GraphPad Prism 9.1.2 (GraphPad Software, Inc.).

| Wavelength sensitivity of C. auris
The results of the wavelength sensitivity tests revealed that 267-270 nm peak wavelengths offered higher disinfection performance (Table 1 and

| Biofilm prevention activity
At 267 nm, UVC LEDs exhibited antibiofilm activity. A dose of 10 mJ cm −2 , when applied at the early phase of biofilm formation, significantly (R 2 = 0.9331, p = 0.0017) inhibited biofilm formation on stainless steel surfaces (Figures 4a and 5a), while completely inhibiting biofilm formation with 30 mJ cm −2 . On plastic, it was possible to prevent biofilm formation with 1 mW cm −2 via static dosing at 10 mJ cm −2 (irradiation 10 s) (Figure 5b). Against poly-cotton fiber, 60 mJ cm −2 significantly reduced biofilm formation (R 2 = 0.9889, p ≤ 0.0001) ( Figure 5c), but was not enough to prevent biofilm formation. These observations could be explained by the surface microstructure differences in the test surfaces ( Figure 5). HAIs . This is likely because C. auris, which is a yeast (unicellular eukaryote), is a higher organism, with seven chromosomes (Muñoz et al., 2021), as opposed to the bacterial strains which mainly have one chromosome (Harrison et al., 2010).

T A B L E 1 UVC efficacy in inactivating
Overall, these results show that C. auris ATCC MYA-5001 is most sensitive to 267-270 nm light and that it requires 20 mJ cm −2 to achieve a 3-log reduction at 267 nm in aqueous. Irradiation at 267 nm with 30 and 10 mJ cm −2 was sufficient to prevent biofilm growth on stainless-steel and plastic coupons, respectively. The significant reduction of biofilm formation on poly-cotton fabric by the study confirms how critical it is for design systems to verify performance against target surfaces. This is because one of the limitations of UVC, like many other disinfectants, is that one solution cannot be applied across the board.

| Regulatory and disinfection considerations
The International Sanitary Supply Association (ISSA) has established cleaning times based on a square foot driven estimate (Insero, 2019). Previous findings have revealed sources of variability in the yeasticidal effect of UVC. One source of variability could be the overlapping of the microbes during disinfection, which could reduce UVC penetrability leading to lower log reduction than expected in real applications (Cevenini et al., 2020). Such overlapping is due to the use of a high concentration of microorganisms during laboratory experiments. Although the sensitivity of the microbial population is not uniform, and thus LRV is not strictly linear with dose, the results from this study imply the prediction from the model by Lemons et al. (2019), which projected that LRV5 (99.999% reduction) can be obtained with 66-110 mJ cm 2 of UVC dose, is likely.

| Effects of test strains and test conditions
It should be emphasized that this study only used one drug-resistant strain from clade II, C. auris type strain (ATCC MYA-5001 = B11220 = JCM 15448 = CBS 10913 = DSM 21092) isolated from the human auditory canal at Tokyo Metropolitan Geriatric Hospital (Satoh et al., 2009). Different strains (for instance, those from clades I, III, IV, and V) may yield slightly different results due to possible differences in sensitivities. Additionally, although temperature and humidity are reported, the effects of temperature and humidity were not explored as the purpose of this study was not to investigate the impact of temperature and humidity on disinfection efficacy. Non-linearities between exposure and disinfection, although known and observed, were not explored in-depth. Also, due to the F I G U R E 3 Disinfection performance against Candida auris versus UVC dose obtained from different arrays. Similar UVC doses for each array were used during testing. Performance of arrays increases with dose. All arrays were designed to deliver the same amount of dose for a similar amount of time to avoid bias. UVC LEDs emitting radiation of 260-270 nm obtained higher disinfection with less time. LED, light-emitting diode; UVC, ultraviolet-C F I G U R E 5 Microstructural differences between (a) stainless-steel, (b) plastic, and (c) poly-cotton fabric coupons used in the biofilm prevention analysis. Biofilm prevention at 267 nm was obtained with 30 and 10 mJ cm −2 on stainless-steel and plastic coupons, respectively. A dose of 60 mJ cm −2 reduced biofilms on poly-cotton fabric significantly (R 2 = 0.9750, p = 0.0002), although was not enough to completely prevent biofilm formation. 8th October 2021). This study did not receive any specific grant from funding agencies in the public or commercial, or not-for-profit sectors.