Journal Pre-proof Validation of methylene blue viability staining with the emerging pathogen Candida auris
Ryan A. Parker, Kyle T. Gabriel, Kayla Graham, Christopher T. Cornelison PII:
S0167-7012(19)30523-8
DOI:
https://doi.org/10.1016/j.mimet.2019.105829
Reference:
MIMET 105829
To appear in:
Journal of Microbiological Methods
Received date:
21 June 2019
Revised date:
13 December 2019
Accepted date:
26 December 2019
Please cite this article as: R.A. Parker, K.T. Gabriel, K. Graham, et al., Validation of methylene blue viability staining with the emerging pathogen Candida auris, Journal of Microbiological Methods (2019), https://doi.org/10.1016/j.mimet.2019.105829
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© 2019 Published by Elsevier.
Journal Pre-proof Validation of methylene blue viability staining with the emerging pathogen Candida auris Ryan A. Parker, Kyle T. Gabriel, Kayla Graham and Christopher T. Cornelison*
[email protected] BioInnovation Laboratory, Kennesaw State University, Kennesaw, GA 30144, USA *
corresponding author at: Kennesaw State University, Mathematics and Statistics Room 232, MD0111, 365 Cobb Avenue, Kennesaw, GA 30144
Abstract
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Methylene blue viability staining has been traditionally used to assess viability of Saccharomyces cerevisiae in brewing and wine making. Here, this method was tested and
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validated with the emerging fungal pathogen Candida auris to determine if this species would also deferentially stain, which could provide utility in assessing microbial control and disinfectant
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efficacy.
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Short Communication
Candida auris is an emerging human fungal pathogen that was first established as a
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distinct species in 2009, after its isolation from the inner ear of a hospital patient in Japan [1]. Since then, unique strains from four different clades have been identified in clinical cases
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worldwide, typically causing nosocomial candidemia which is often lethal [2]. Complicating intervention efforts, C. auris is often misidentified when using traditional diagnostic methods [2].
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Furthermore, C. auris has been shown to survive for weeks on soiled surfaces and be tolerant of temperatures as high as 40 °C [3]. Many strains are also multi-drug resistant, with some even
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being found resistant to all three major classes of antifungal agents [4]. C. auris has been documented to persist on fomites, which could pose as a possible reservoir for the pathogen. This could allow transmission to vulnerable patients through contact with these contaminated surfaces. As researchers race to discover effective methods for controlling C. auris, the ability to assess viability, or the amount of living cells in a culture, in a timely manner becomes ever more important. A stain that differentiates viable cells would provide a rapid method to assess the load of viable cells during disinfection testing. Currently, viability assessments are conducted using serial dilution and plate count methods. While these methods are effective at determining viability, it is time- and resource-intensive, as well as prone to issues with contamination. Due to incubation requirements, viability based on plate counts cannot be determined in less than 48 hours. In the case of C. auris, this is further complicated by the lack of organism-specific standardized methods for determining disinfectant efficacy. To
Journal Pre-proof illustrate this, the Environmental Protection Agency has only recently established guidelines for testing potential disinfectants with C. auris, and the Centers for Disease Control and Prevention (CDC) has suggested the use of chemicals approved for use with Clostridium difficile to eliminate C. auris from surfaces [5]. A hemocytometer allows for quick and accurate enumeration of cells, but without additional tools, it is impossible to determine viability. In the field of brewing, methylene blue staining is used to quantify the viability of Saccharomyces cerevisiae used in alcohol fermentation. This allows a brewer to determine the amount of culture to add to the fermentation
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vessel that will contain an appropriate number of living cells [6]. In metabolically-active cells, methylene blue is metabolized to a colorless compound and the cells retain their hyaline
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appearance under microscopic evaluation. In metabolically-inactive (i.e. nonviable) cells, the stain remains, and the cells appear blue. This investigation sought to test the efficacy of staining
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C. auris to potentially combine cell counting with viability determination.
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C. auris (AR0389) of the CDC Candida auris panel was used as the challenge organism and S. cerevisiae (AR0399) from the same panel was used as the control organism. The
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organisms were received as glycerol stocks, which were inoculated to malt extract broth (MEB) and malt extract agar (MEA). All cultures were incubated at 37 °C for two days prior to assaying.
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Samples from plate colonies of each organism were used to create glycerol stocks for long-term storage at −80 °C. The plates were retained for future use and preserved by wrapping in
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paraffin film and storing at 4 °C. Liquid cultures of S. cerevisiae and C. auris were grown in MEB at 37 °C with 250 RPM shaking. Each liquid culture was divided into two equal samples. One of
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the samples was heated to 100 °C for 10 minutes while the other was held at room temperature, generating four experimental groups. A 100 μL aliquot from each sample was mixed with an equal volume of 0.02% methylene blue and incubated for 5 minutes. Each stained sample was loaded into a hemocytometer (Bright-Line 400180, Reichert Technologies, New York, USA) and microscopically observed to enumerate and determine the percentage of viable cells. Viable cells were identified by the retention of their normal hyaline appearance. To validate the viable counts estimated with the hemocytometer and methylene blue, the experiment was repeated with the addition of the following step. Samples from the treatment groups were diluted to less than 103 cells mL-1, viable or otherwise, using the hemocytometer. From the diluted samples, 0.1 ml of each were spread across the surface of MEA plates and incubated at 37 °C for 48 hours. Aliquots of treatment and control were also blended a volumetric ratios to evaluate the All trials were performed in triplicate. The hemocytometer and plate counts were compared to
Journal Pre-proof determine the accuracy of the staining method to quantify viable cells. A two-tailed t-test, assuming unequal variance, was used to determine if any statistically significant difference was present between the two methods. Following staining with methylene blue, the boiled samples of both organisms retained the blue coloration typical of nonviable cells (Figure 1B and 1D), while the non-boiled samples
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remained hyaline (Figure 1A and 1C).
Fig. 1. Hemocytometer grid at 400x magnification with viable, non-viable, and blended Saccharomyces cerevisiae and Candida auris after being stained with methylene blue. S. cerevisiae 100% viable (A), S. cerevisiae 100% non-viable (B), S. cerevisiae 75% viable blend (C), C. auris 100% viable (D), C. auris 100% non-viable (E) and C. auris 75% viable blend. Nonviable cells are stained blue. Smaller squares, outlined by single lines, are 0.0025 mm 2. There was no significant difference (p ≥ 0.99) in viability between hemocytometer and plate count methods (Figure 2). No viability was detected from the boiled samples using either method and plate count and viability quantification of both methods was consistent across blended sample ratios (Figure 2).
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Viable Cells ml -1
1.00E+06
1.00E+05 1.00E+04 1.00E+03
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1.00E+02
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1.00E+01
Ca 3:1 viable
Ca 1:1 viable
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1.00E+00
Ca 1:3 viable
Sc 1:1 viable
Sc 1:3 viable
Plate Counts
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Methylene Blue Viability
Sc 3:1 viable
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Fig. 2. Viability of Candida auris and Saccharomyces cerevisiae using hemocytometer enumeration following methylene blue staining and traditional plate counts. Results are the
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average of triplicate counts for each, with the average being displayed above the corresponding bar. Units presented are viable cells for hemocytometer counts and colony-forming units for
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plate counts.
Methylene blue viability staining was successful in differentially staining C. auris and the
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resulting viability assessment was statistically consistent with the serial dilution and plate count methodology. This staining method is simple, inexpensive, time-efficient, and could easily be applied to many antimicrobial testing methods where assessing viability is desired. A plate count requires two to three days to obtain confident results, due to the long growth periods required by C. auris. Using methylene blue and a hemocytometer, equivalent results were obtained in less than an hour. Because this stain has worked on two different yeast species, it is also reasonable that this method could be extended to other Candida species and possibly even other pathogenic yeast. This method does have limitations, as hemocytometers typically have a lower limit of detection, at 104 cells mL-1, which could preclude its use if the starting inoculum density is below this threshold. However, centrifugation may be utilized to overcome this limitation.
Journal Pre-proof New multi-drug-resistant C. auris isolates continue to be identified. This, coupled with many other fungal species developing resistance to a variety of treatment drugs, underscores the need for production of novel antifungals and faster testing methods. Methylene blue provides a low-cost and rapid method for quantifying C. auris viability. This method may be able to significantly increase the efficiency and accuracy of testing antimicrobials for managing C. auris. Author Statement –
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Ryan Parker: Investigation, formal analysis, writing. Kayla Graham: investigation, formal analysis. Kyle Gabriel: Validation, methodology, formal analysis. Chris Cornelison: Conceptualization, methodology, writing, project administration, funding acquisition.
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Declaration of interests: None
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References
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1. Satoh K, Makimura K, Hasumi Y, Nishiyama Y, Uchida K, Yamaguchi H. 2009. Candida auris sp. nov., a novel ascomycetous yeast isolated from the external ear canal of an inpatient in a Japanese hospital. Microbiol Immunol. 53(1):41-4. 2. Jeffery-Smith A, Taori S, Schelenz S, Jeffery K, Johnson E, Borman A, Candida auris Incident Management Team, Manuel R, Brown C. 2017. Candida auris: A review of the literature. Clin Microbiol Rev. 31(1):17-35. 3. Welsh R, Bentz M, Litvintseva A, Shams A, Houston H, Lyons A, Rose L. 2017. Survival, persistence, and isolation of the emerging multidrug-resistant pathogenic yeast Candida auris on a plastic health care surface. J Clin Microbiol. 55(10):2996-3005. 4. Sarma S, Upadhyay S. 2017. Current perspective on emergence, diagnosis and drug resistance in Candida auris. Infection and Drug Resistance, 10:155-65 5. Interim Guidance for the Efficacy Evaluation of Products for Claims against Candida auris. 2017 May 24. EPA. https://www.epa.gov/pesticide-registration/interim-guidanceefficacy-evaluation-products-claims-against-candida-auris-0 6. Painting K & Kirsop B (1990) A quick method for estimating the percentage of viable cells in a yeast population, using methylene-blue staining. World J Microbiol Biotechnol 6: 346–7.
Highlights
Candida auris is an emerging human fungal pathogen. Methylene blue viability assessment was evaluated with Candida auris. Determine viability of Candida auris for antimicrobial efficacy assessment. This method significantly reduces the time necessary to determine viability.