The emerging pathogenic multidrug-resistant yeast Candida auris is an important source of healthcare-associated infections and of growing global clinical concern. The ability of this organism to survive on surfaces and withstand environmental stressors creates a challenge for eradicating it from hospitals. A panel of C. auris clinical isolates was evaluated on different surface environments against the standard disinfectant sodium hypochlorite and high-level disinfectant peracetic acid. C. auris was shown to selectively tolerate clinically relevant concentrations of sodium hypochlorite and peracetic acid in a surface-dependent manner, which may explain its ability to successfully persist within the hospital environment

Akinbobola, A.

Jones, B.L.

Kean, R.

MacKay, W.G.

McKloud, E.

Ramage, G.

Sherry, L.

Short, B.

Townsend, E.

Williams, C.

Journal of Hospital Infection

English

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Surface disinfection challenges for Candida auris: an in-vitro study

The emerging pathogenic multidrug-resistant yeast Candida auris is an important source of healthcare-associated infections and of growing global clinical concern. The ability of this organism to survive on surfaces and withstand environmental stressors creates a challenge for eradicating it from hospitals. A panel of C. auris clinical isolates was evaluated on different surface environments against the standard disinfectant sodium hypochlorite and high level disinfectant peracetic acid. C. auris was shown to selectively tolerate clinically relevant concentrations of sodium hypochlorite and peracetic acid in a surface dependent manner, which may explain its ability to successfully persist within the hospital environment

Kean, Ryan

Sherry, Leighann

Townsend, Eleanor

McKloud, Emily

Short, Bryn

Akinbobola, Ayorinde

Mackay, William G.

Williams, Craig

Jones, Brian L.

Ramage, Gordon

Research Repository and Portal - University of the West of Scotland

Surface disinfection challenges for <em>Candida auris</em>:an in vitro study

R. Kean

L. Sherry

E. Townsend

E. McKloud

B. Short

A. Akinbobola

W.G. Mackay

C. Williams

B.L. Jones

G. Ramage

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 1 Surface disinfection challenges for Candida auris: an in vitro study 1  2 Ryan Kean1,2, Leighann Sherry1, Eleanor Townsend1, Emily McKloud1, Bryn 3 Short1, Ayorinde Akinbobola2, William G Mackay2, Craig Williams2, Brian L 4 Jones3 and Gordon Ramage1* 5  6  7  8 1School of Medicine, Dentistry and Nursing, College of Medical, Veterinary and 9 Life Sciences, Glasgow; 2Institute of Healthcare Policy and Practice, School of 10 Health, Nursing, and Midwifery, University of the West of Scotland, Paisley; 11 3Microbiology Department, Glasgow Royal Infirmary, NHS Greater Glasgow 12 and Clyde, Glasgow UK. 13  14  15 Running title: Surface disinfection challenges for Candida auris 16  17 Key words: Candida auris, disinfection, surface 18  19  20 *Corresponding Author: Gordon Ramage, Oral Sciences Research Group, 21 Glasgow Dental School, School of Medicine, Dentistry and Nursing, College of 22 Medical, Veterinary and Life Sciences, University of Glasgow, 378 Sauchiehall 23 Street, Glasgow, G2 3JZ, UK. Phone: +44(0)141 211 9752. e-mail: 24 gordon.ramage@glasgow.ac.uk 25   26  2 Abstract 1 The emerging pathogenic multidrug-resistant yeast Candida auris is an 2 important source of healthcare–associated infections and of growing global 3 clinical concern. The ability of this organism to survive on surfaces and 4 withstand environmental stressors creates a challenge for eradicating it from 5 hospitals. A panel of C. auris clinical isolates was evaluated on different 6 surface environments against the standard disinfectant sodium hypochlorite 7 and high level disinfectant peracetic acid. C. auris was shown to selectively 8 tolerate clinically relevant concentrations of sodium hypochlorite and peracetic 9 acid in a surface dependent manner, which may explain its ability to 10 successfully persist within the hospital environment.  11  12  13   14  3 Introduction 1 Fungal infections affect more than a billion people, resulting in approximately 2 11·5 million life-threatening infections and more than 1.5 million deaths 3 annually. There have been significant strides made in tackling these infections 4 over the past decade, but the global impact of these measures has yet to be 5 realized [1]. An important fungus worth consideration in this context is the 6 multidrug-resistant yeast Candida auris, which has been increasingly 7 described as a major global concern and cause of major nosocomial outbreaks 8 [2]. The impact on transmission and infection control is substantial, so 9 understanding their mechanisms of spread and survival in the hospital 10 environment is critical, particularly as it is able to persist on plastics and steel, 11 and survive as biofilms [3, 4]. Several recent investigations have established 12 that C. auris is capable of prolonged survival on surfaces [4, 5], and that 13 surface disinfection protocols had variable and unsatisfactory outcomes [5]. 14 Given that it has been shown recently that 1000ppm of an active chlorine 15 solution is highly effective against these organisms tested in suspension [6], 16 then it is clear that surfaces play an important role in survival of this pathogen. 17 Our own work confirms this, with C. auris biofilms being generally insensitive to 18 a range of key antimicrobial agents, thus prolonging their survival capacity [3]. 19 Therefore, identifying ways to minimise the impact of C. auris within the 20 hospital environment is imperative. The purpose of this study was to 21 investigate the general disinfectant sodium hypochlorite (NaOCl), commonly 22 used for terminal cleaning within the hospital environment, and the high level 23 disinfection agent peracetic acid (PA), on different substrate surfaces. These 24 data will support our understanding of how C. auris responds to different levels 25 of challenge on surfaces representative of the hospital environment. 26  27 Material and Methods 28 Strains and culture conditions 29 Throughout this study four Candida auris (Ca) isolates obtained from various 30 clinical sites [7], (NCPF 8971, NCPF 8973, NCPF 8977, NCPF 8978) were 31 used, as previously described [3]. All isolates were identified by ribosomal DNA 32 (rDNA) gene sequencing or matrix-assisted laser desorption ionization–time of 33 flight (MALDI-TOF) [7]. Candida glabrata (Cg) ATCC 2001 and Candida 34  4 albicans (Ca) ATCC 10231 was used as reference strains. All strains were 1 stored and maintained on Sabouraud dextrose (SAB) agar (Oxoid, Hampshire, 2 UK) prior to propagation in yeast peptone dextrose (YPD) (Sigma-Aldrich, 3 Dorset, UK) medium overnight at 30°C. Cells were prepared according to a 4 modified version of the British Standards for chemical disinfectants and 5 antiseptics [8]. Briefly, cells were washed by centrifugation in phosphate 6 buffered saline ([PBS] Sigma-Aldrich, Dorset, UK), and standardised to 1 x 107 7 cells/mL in sterile water containing 5% foetal bovine serum to simulate organic 8 material.  9  10 Surface disinfection testing 11 The following test surface substrates were used: cellulose matrix (IPS 12 Converters, Oldham, UK [1.25cm2]), 304 stainless steel (LaserMaster, 13 Redruth, UK [3.14cm2]) and Thermanox™ polyester coverslips (Fisher 14 Scientific, Loughborough, UK [1.32cm2]). Following the adhesion phase, non-15 adherent cells were removed by washing with 1 mL PBS.  Next, each surface 16 was challenged with either NaOCl (1000 and 10000 ppm, [Fisher Scientific, 17 Loughborough, UK]) or PA (2000ppm [Acros Organics, Geel, Belgium]), both 18 agents diluted to their respective working concentrations in sterile water. 19 Following 5 min or 10 min exposure, disinfectants were neutralised with 5% 20 sodium thiosulphate (Fisher Scientific, Loughborough, UK) for 15 min. The 21 neutraliser alone did not have a detrimental impact on Candida viability when 22 treated in the absence of a disinfectant (data not shown). Substrate sections 23 were then sonicated at 35 kHz for 10 min in sterile H2O to remove cells, and 24 serial ten-fold dilutions in sterile water were plated on to SAB agar according to 25 the Miles and Misra plate count method. These were then incubated at 30°C 26 for 48 h. Parallel experiments were also performed to assess the potential for 27 regrowth following disinfection procedures. After treatment and neutralisation 28 as described above, test coupons were replaced in 10mL of fresh YPD media 29 and incubated for 24 h at 30°C with gentle of 100 rpm. Substrate adhered C. 30 auris cells treated with sterile water acted as a positive control, with substrates 31 containing no C. auris cells included as negative controls throughout this study. 32 After 24 h, the optical density readings were measured at a wavelength of 33  5 530nm (OD530) using a microtitre plate reader (FluoStar Omega, BMG 1 Labtech, Aylesbury, UK).  2  3 Statistical analysis 4 Data distribution, statistical analysis and graph production was performed 5 using GraphPad Prism (version 7; La Jolla, CA, USA). Student t-tests were 6 used to compare treated and untreated samples. A one-way analysis of 7 variance and post-hoc Tukey test was used to compare the effectiveness of 8 each disinfectant against the 3 different substrates. All experiments were 9 performed in triplicate on three independent substrates, with the mean of each 10 experiment used for analyses. Statistical significance was achieved if p<0.05. 11  12 Results  13 Initially, a standard disinfectant challenge was performed against C. auris on 14 different substrates relevant to the hospital environment. A cellulose substrate 15 was included to act as control for porosity. It was shown that all four C. auris 16 were significantly killed by NaOCl challenge at 1000 and 10000ppm, 17 irrespective of substrate and strain, though differences were observed between 18 these substrates. Complete eradication (100%) was only achieved on the 19 cellulose substrate (Fig 1A). On the non-porous materials, significant quantities 20 of viable yeast cells were killed on the steel surface following NaOCl at all 21 treatment parameters, with an approximate 2.5 log10 reduction (p<0.001), with 22 no significant differences observed at each time point and concentration tested 23 (Fig 1B). Notably, those isolates treated with 1000ppm for 5 min showed 24 significantly more regrowth compared to the other test conditions (p<0.001). 25 When C. auris was tested on a polymer substrate it was shown that 5 min 26 exposure at 1000ppm was the least effective overall, and although there was 27 significant activity observed (mean log10 reduction = 1.29; p<0.001), 4.95 log10 28 was retained on the surface (Fig 1C). However, following an increased contact 29 time of 10 min or increased concentration of 10000ppm, significantly enhanced 30 activity was observed compared to the 5 min contact time (p<0.001), with an 31 approximate overall 3.5 log10 reduction. When comparing both increased 32 treatment parameters, no significant differences were observed between the 33 regimens (P=0.347), and no notable regrowth was detected. 34  6  1 Following a standard disinfection challenge, the efficacy of the HDL agent PA 2 was assessed. When tested against 2000ppm of PA, it was shown that all C. 3 auris isolates were significantly killed by this agent. However, differences were 4 again detected between substrates. As observed with NaOCl, complete 5 eradication (100%) was achieved on the cellulose matrix (Fig 2A), with this 6 same fungicidal activity also observed on the polymer substrate (Fig 2B). 7 However, compared to the other two substrates, significant quantities of viable 8 cells were recovered from the steel substrate following PA challenge (mean 9 log10 = 3.19; p<0.001), with an overall 2.70 log10 reduction (p<0.001) (Fig 2C). 10 When re-inoculated into media post-challenge, substantial regrowth was 11 recorded from both steel and polymer substrates, with minimal quantities 12 recovered from the cellulose substrate. 13  14 For both disinfectants on each of the substrates, no differences were observed 15 between strains, and both exhibited a similar profile to C. glabrata and C. 16 albicans. Similarly, the presence of BSA was shown to have no effect of any 17 treatments compared to no BSA controls. Liquid suspension tests showed that 18 NaOCl and PA were highly effective at <20ppm and 40ppm, respectively. 19  20 Discussion 21 Although the precise mechanism of C. auris nosocomial transmission remains 22 unknown, it is thought to be a multi-factorial process whereby it can colonise 23 the environment and equipment of the healthcare setting. It has been reported 24 to tolerate a number of environmental stressors, including temperature and 25 salt, and some strains appear to have variable aggregative phenotypes that 26 may have a role in persistence within the environment and the host [7, 9]. We 27 therefore set out to investigate how resilient C. auris is within a controlled 28 disinfection challenge using clinical isolates from the UK [7]. Here we report for 29 the first time that both standard and high-level disinfection strategies were 30 unable to completely eradicate C. auris from non-porous substrates.  31  32 Chlorine based disinfectants have variable yeasticidal activity against 33 planktonic C. auris [6, 10], though their role in surface disinfection procedures 34  7 lacks definitive evidence. Recently, it has been shown that quaternary 1 ammonium compounds were poorly active against C. auris, whereas 2 environmental protection agency registered hospital disinfectants, such as 3 NaOCl containing solutions, were fungicidal on surfaces [5]. In a recent UK 4 outbreak, Schelenz and colleagues (2016) implemented chlorine based 5 disinfectants at 1000ppm three times daily for environmental cleaning, and 6 10000ppm for terminal cleaning [2]. The data presented herein support this 7 clinical guidance, though length of exposure at 1000ppm or an increased 8 concentration is an important factor for consideration to maximize C. auris 9 control. This is evident from our analysis of recovery of cultivable cells directly 10 following surface challenge at 1000 ppm for 5 min, which suggests this is an 11 ineffective control strategy. 12  13 It was interesting to note that we observed a significant difference in activity 14 when comparing the polymer to steel, which could be explained by Candida 15 species general ability to adhere and form biofilm, which are inherently more 16 resistant. This is reinforced by another study investigating these responses on 17 steel, where different exposure times and concentrations were reported to 18 effectively reduce C. auris viability by up to 6 log10 [5]. Taken together, these 19 data suggest the standard disinfection procedures are surface dependent, and 20 given the diversity of fomites in the hospital setting then this could pose a 21 problem for disinfection. To this end we decided to explore a representative 22 high-level disinfection protocol. Here PA was used, a disinfectant routinely 23 used for endoscope reprocessing. Contrary to our previous data, it was shown 24 that on plastic polymers this disinfectant challenge was more effective, 25 showing significant reduction compared to stainless steel. This is the first 26 report to investigate this agent, and although it was used to represent a 27 superior disinfectant strategy, it revealed a risk for potential transmission via 28 contaminated endoscopes. C. auris has been isolated from a number of clinical 29 sources [9], so it is not unreasonable to suggest that this and other hospital 30 instruments could facilitate transmission.   31  32 Recent studies have suggested that C. auris has been shown to survive on 33 steel and plastic surfaces for 1 and 4 weeks, respectively [4, 11]. Comparison 34  8 of C. auris to C. parapsilosis persistence on plastics was quantified under 1 controlled hospital conditions (temperature and humidity). Low density test 2 suspensions (104) of C. auris was shown to remain viable (CFU counts) for up 3 to 14 days, though more sensitive esterase measurements suggested viable 4 activity up to 28 days that was comparable to C. parapsilosis [4]. Piedrahita 5 and colleagues (2017) further investigated C. auris in comparison to C. 6 albicans, C. glabrata and C. parapsilosis, specifically looking at moist and dry 7 inoculums over 7 days. Here they demonstrated that in moist conditions all 8 species were recovered in near maximum efficiency after 7 days, whereas only 9 40% of the dried inoculum were recovered on the steel substrates, which was 10 significantly greater than C. albicans, though C. glabrata and C. parapsilosis 11 were recovered by approximately 65% [11]. This is in line with our own 12 findings, showing comparable disinfectant sensitivity profiles for both C. auris 13 and C. glabrata. Nevertheless, given the multi-drug resistance phenotype of C. 14 auris compared to other species then its ability to persist is particularly 15 alarming.  16  17 While this study provides a useful insight into potential complications with 18 disinfectant procedures, there are some limitations. We have only been able to 19 test a limited panel of C. auris strains, although they did demonstrate similar 20 sensitivity profiles across each tested parameter. Furthermore, we only studied 21 two disinfectants at individual concentrations and contact times. Future studies 22 aim to undertake extensive analysis with commercial products in conjunction 23 with up-to-date infection control guidelines. Overall, this study reveals the 24 potential deficits we have in controlling this emerging fungal pathogen, and 25 only through understanding the biology of this multi-resistant pathogen will 26 assist us in devising new therapeutic and control interventions.    27  28 Acknowledgements 29 We would like to thank the European Society for Clinical Microbiology and 30 Infectious Disease for financial support for LS.  31  9 References 1 [1] Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC 2 Hidden killers: human fungal infections. Sci Transl Med 2012; 4: 3 165rv13. 4 [2] Schelenz S, Hagen F, Rhodes JL et al. First hospital outbreak of the 5 globally emerging Candida auris in a European hospital. Antimicrob 6 Resist Infect Control 2016; 5: 35. 7 [3] Sherry L, Ramage G, Kean R et al. Biofilm-Forming Capability of Highly 8 Virulent, Multidrug-Resistant Candida auris. Emerg Infect Dis 2017; 23: 9 328-31. 10 [4] Welsh RM, Bentz ML, Shams A et al. Survival, Persistence, and 11 Isolation of the Emerging Multidrug-Resistant Pathogenic Yeast 12 Candida auris on a Plastic Healthcare Surface. J Clin Microbiol 2017; 13 doi 10.1128/JCM.00921-17. 14 [5] Cadnum JL, Shaikh AA, Piedrahita CT et al. Effectiveness of 15 Disinfectants Against Candida auris and Other Candida Species. Infect 16 Control Hosp Epidemiol 2017; doi 10.1017/ice.2017.162: 1-4. 17 [6] Moore G, Schelenz S, Borman AM, Johnson EM, Brown CS Yeasticidal 18 activity of chemical disinfectants and antiseptics against Candida auris. 19 J Hosp Infect 2017; doi 10.1016/j.jhin.2017.08.019. 20 [7] Borman AM, Szekely A, Johnson EM Comparative Pathogenicity of 21 United Kingdom Isolates of the Emerging Pathogen Candida auris and 22 Other Key Pathogenic Candida Species. mSphere 2016; 1. 23 [8] BSI. Chemical disinfectants and antiseptics - Quantitative test method 24 for the evaluation of bactericidal and yeasticidal activity on nonporous 25 surfaces with mechanical action employing wipes in the medical area 26 (4- field test) - Test method and requirements  (phase 2, step 2).  BS EN 27 16615:2015,  London: BSI Standards Publication 2015. 28 [9] Chowdhary A, Sharma C, Meis JF Candida auris: A rapidly emerging 29 cause of hospital-acquired multidrug-resistant fungal infections globally. 30 PLoS Pathog 2017; 13: e1006290. 31 [10] Abdolrasouli A, Armstrong-James D, Ryan L, Schelenz S In vitro 32 efficacy of disinfectants utilised for skin decolonisation and 33 environmental decontamination during a hospital outbreak with Candida 34 auris. Mycoses 2017; 60: 758-63. 35 [11] Piedrahita CT, Cadnum JL, Jencson AL, Shaikh AA, Ghannoum MA, 36 Donskey CJ Environmental Surfaces in Healthcare Facilities are a 37 Potential Source for Transmission of Candida auris and Other Candida 38 Species. Infect Control Hosp Epidemiol 2017; doi 10.1017/ice.2017.127: 39 1-3. 40  41  10  1  2 Figure 1. Efficacy of sodium hypochlorite on Candida auris, Candida glabrata 3 and Candida albicans on three different substrates. 4   5 Cellulose matrix (A), stainless steel (B) and polymer (C) were inoculated with 1 6 x 107 cells/mL of C. auris (NCPF), C. glabrata (CG) and C. albicans (CA) for 90 7 min before being treated with 1000ppm NaOCl for 5 min, 1000ppm for 10 min 8 and 10000ppm for 5 min. Viable cells were then quantified by CFU (left hand y-9 axis) and regrowth was measured spectrophotometrically (right hand y-axis). 10 Data represents means ± standard deviation of triplicate datasets, with CFU 11 log10 reduction of each test substrate normalised to 1cm2.  # indicates 12 complete eradication compared to untreated control. N.A. = not applicable. 13  14  15 Figure 2. Efficacy of PA on Candida auris, Candida glabrata and Candida 16 albicans on three different substrates. 17   18 Cellulose matrix (A), stainless steel (B) and polymer (C) were inoculated with 1 19 x 107 cells/mL of C. auris (NCPF), C. glabrata (CG) and C. albicans (CA) for 90 20 min before being treated with 2000ppm PA for 5 min. Cell viability (left hand y-21 axis) and re-growth (right hand y-axis) were quantified by CFU and 22 spectrophotometric readings, respectively. Data represents means ± standard 23 deviation of triplicate datasets, with CFU log10 reduction of each test substrate 24 normalised to 1cm2. # indicates complete eradication compared to untreated 25 control.  26 

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Surface disinfection challenges for Candida auris: an in-vitro study

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