Does repeated exposure to hydrogen peroxide induce Candida auris resistance?

Background To minimize environmental colonization by microorganisms that may persist and thrive in healthcare settings, thus reducing healthcare-associated infections (HAIs), new insights over already known biocides are certainly of relevance. Although the efficacy of hydrogen peroxide (H2O2) against the emergent yeast Candida auris is moderately documented, concerns over the potential induction of resistance after repeated exposure do persist. The main objective of the present study was to evaluate the hypothetical induction of Candida auris resistance following 30 days of consecutive exposure to lethal and sublethal concentrations of H2O2. Furthermore, the authors aimed to elucidate about the rank of efficacy of H2O2 against C. auris comparing to other Candida species and whether different strains of C. auris may display different susceptibilities to H2O2. Methods During the induction of resistance assays, both type strains and clinical isolates of Candida auris, Candida albicans and Candida parapsilosis were exposed repeatedly to defined concentrations of H2O2, for 30 days. Results After that period, no significant differences were found when comparing the minimal inhibitory concentration values of H2O2 in case of the induced strains versus each respective positive control. Moreover, H2O2 displayed similar effectiveness against all the tested Candida species and no differences were demonstrated among the distinct strains of C. auris. Conclusions The adoption of H2O2 solutions in routine protocols in order to promote disinfection standards against Candida auris, improving patient safety and reducing healthcare costs, is certainly welcomed.


Background
Presently, quite optimistic results regarding the prevention of healthcare-associated infections (HAIs) are being made available in the literature.However, the daily efforts of healthcare workers and further consistent scientific research are needed to fuel that trend.To minimize the colonization by infectious microorganisms able to persist and thrive in the healthcare environment, new strategies for cleaning and disinfection are being proposed, but with variable success [1][2][3].Yet, no matter how appealing the new methods and biocide candidates may sound, new insights over already known and extensively used biocides are certainly welcomed and may play a relevant role towards the effective prevention of HAIs.
Candida auris is an emerging pathogen that has been isolated in several countries in a relatively short period of time, causing multiple outbreaks [4][5][6][7][8][9][10][11].Even though both the aggregative and the non-aggregative phenotypic variants are able to form biofilm, the non-aggregative cell type has been more frequently isolated from infected patients, forming a more robust biofilm and displaying higher virulence [12][13][14][15].C. auris has been associated with multidrug resistance, severe infections and high mortality rates [6,8,16].Clinical isolates have been recovered most frequently from the blood of critically ill patients with candidemia, including cases associated with central venous catheter use, but also from patients with respiratory tract and urinary tract infections [5,8,9,17].Although several reports regarding nosocomial transmission have been published [8,10,16], data are still insufficient concerning the effectiveness of disinfectants against C. auris [18].
Currently, hydrogen peroxide (H 2 O 2 ) is extensively used in healthcare settings, either as a liquid agent for surface disinfection, or as a vapour or aerosol for terminal disinfection and sterilization.The biocide action of H 2 O 2 occurs through the generation of hydroxyl free radicals that damage essential cell components such as lipids, proteins and DNA, exhibiting a broad-spectrum activity against diverse bacteria, fungi and viruses [19][20][21].However, the level of evidence of H 2 O 2 efficacy against C. auris remains moderate [22] and, as far as it is known to the authors, no data exists addressing the potential induction of resistance after repeated use of H 2 O 2 .
Therefore, the main objective of the present study was to evaluate the potential induction of C. auris resistance following 30

Microbial strains and culture conditions
Details about the strains used in the study are displayed in Table 1.Candida albicans CA 016 and Candida parapsilosis CP 009 clinical strains were obtained from the strain collection of the Microbiology Division, Department of Pathology of the Faculty of Medicine, University of Porto, Portugal.Such strains were previously recovered from critical care patients.They were kept at -80ºC, in Brain Heart infusion broth with glycerol.Identification of clinical isolates was initially performed with Vitek system (bioMérieux, Vercieux, France) and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, Bruker).
Candida Following thawing, the yeast strains were initially maintained in Sabouraud Dextrose Agar (Liofilchem, Italy), at 37 °C.For growth in liquid media, YPD (Yeast Peptone Dextrose broth medium, Liofilchem, Italy) was used; strains were cultivated at 37 °C, 120 rpm, constant temperature in an incubator shaker.

H 2 O 2 susceptibility testing
Initial (day 0) minimal inhibitory concentrations (MICs) of H 2 O 2 (Labkem, Barcelona, Spain) were determined for all strains (Table 1), adapted from protocols M27-A3 and supplement M27-S4 by the Clinical Laboratory for Standards Institute (CLSI) [23][24][25].Briefly, a stock solution of H 2 O 2 (30%) was used to prepare serial two-fold dilutions of H 2 O 2 solutions in RPMI 1640 (Sigma -Aldrich, St Louis, USA) and the final concentrations tested ranged from 0.875 to 0.00171%.Inocula were prepared from 24 h cultures, according to the same protocols, i.e., a stock suspension was initially prepared with a concentration of 1 × 10 6 to 5 × 10 6 cells per mL, corresponding to a 0.5 McFarland standard.Then, a working suspension was prepared by a 1:100 dilution followed by a 1:20 dilution of the stock suspension with RPMI 1640 broth medium, which resulted in 5.0 × 10 2 to 2.5 × 10 3 cells per mL.MICs were determined in 96-well plates, adding to each well 100 µl of the final inoculum and 100 µl of the respective serial dilution of the H 2 O 2 solution.Positive control wells containing only inoculum and RPMI 1640 medium and negative control wells containing the lowest concentration of H 2 O 2 solution and RPMI 1640 medium were included.For each strain, 2 replicates were performed for the determination of MIC values of hydroxide peroxide,  2).Every 24 h, 1 mL of each culture was resuspended into 9 ml of fresh medium containing the same concentration of H 2 O 2 (along with the respective positive control), for a 30-day period.From each daily sub-culture, an aliquot was stored at -80 °C in BHI with 40% glycerol.Every 2 days, a 10 µl loopful of yeast cells was cultured in Sabouraud agar plates to check for culture contamination.Every 10 days along the 30 day duration of the assay, H 2 O 2 MIC was determined according to CLSI protocols [23][24][25].During the induction period, strain identification was confirmed at days 10, 20 and 30 of incubation by MALDI -TOF.

Induction of resistance to H 2 O 2 assessment
The MICs of H 2 O 2 corresponding to the induced strains and their controls are detailed in

Discussion
The search for more efficient protocols to clean and disinfect surfaces of healthcare settings is constant and certainly driven by novel methods and technology, with much relevant published work on the subject [1,2,[26][27][28][29][30][31].However, much more scarcely do novel substances with disinfectant claims arise in the market.Therefore, new insights over extensively used disinfectants are mostly welcomed.
The test method chosen to study the exposure of pathogenic yeasts to H 2 O 2 for a long period of time has obvious limitations, such as the underestimation of the real MICs since H 2 O 2 is an oxidizing agent stable in solution, but unstable in the presence of organic molecules such as those present in the media used for determining the MICs.Besides, differences in the MIC values were smaller than expected because no dramatic genetic shifts were expected to occur by the action of H 2 O 2 .Taken all this together, the most relevant points of the study will be highlighted.
Firstly, even though H 2 O 2 has demonstrated to be microbicidal against several bacteria, viruses, yeasts and spores, its activity against different strains of C. auris and concerns about the potential induction of microbial resistance after repeated exposure do subsist.Since yeasts may persist in the environment for weeks to months [32], most certainly due to ineffective cleaning and disinfection protocols and to the emergence of resistance to biocides, the current study addressed the potential of Candida organisms to develop resistance following prolonged exposure.The authors tested Candida spp frequently associated to HAIs, many of them with a high propensity to colonize hospital settings and persist in the environment for a long time, as is the case with C. parapsilosis and C. auris.MIC values ranged between 0.007% and 0.055%, which are considerably lower than the concentrations of solutions of H 2 O 2 used in disinfection in real healthcare settings.Moreover, during the 30 days of the experiment, non-significant differences were found between MICs of the induced strains and their respective positive controls.
Secondly, regardless C. auris had been previously described as displaying different resistance to H 2 O 2 in comparison to C. albicans [33][34][35], such was not the case in this study.In fact, a similar resistance pattern was found for the three yeast species, as demonstrated by the non-significant differences in MIC values.
Finally, in contrast with a previous publication [36], the effectiveness of H 2 O 2 against C. auris did not differ significantly depending on the C. auris strains (Table 2).

Conclusions
H 2 O 2 did not induce yeast resistance during the test period, in particular regarding the emergent and alarming C. auris (either aggregative or non-aggregative strains), which is undoubtedly good news for a biocide with such an extensive use in healthcare scenarios.The adoption of H 2 O 2 solutions in routine protocols to improve disinfection standards, promoting patient safety and reducing healthcare costs, is certainly highly welcomed.
albicans ATCC 90,028 and Candida parapsilosis ATCC 22,019, from the American Type Culture Collection, were also used in the experiments.C. auris DSMZ 21,092 was obtained from the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH.C. auris NCPF 8971 and C. auris NCPF 8977 were obtained from the Culture Collections Public Health England, National Collection of Pathogenic Fungi.

Table 1
Details about the strains used in the study and this determination was performed twice in different days.Results were read after 24 and 48 h of incubation at 35ºC.MIC values were defined as the lowest concentration of H 2 O 2 which inhibited microbial growth > 99%.

Table 2 .
After 30 days of incubation with H 2 O 2 , MIC values ranged between 0.007% and 0.055% for all strains.In the vast majority, no difference was found between MICs of H 2 O 2 solution in the case of the induced strain and its respective positive control.In a minority of cases (C.parapsilosis CP 009 and C. auris NCPF 8971), at day 30, a non-significant difference of 1 single dilution was found between MIC of H 2 O 2 solution in the case of the induced strain versus its respective positive control.

Table 2
MICs of H 2 O 2 (%) corresponding to the induced strains of yeasts and respective controls