Medical Mycology, 2014, 52, 766–769 doi: 10.1093/mmy/myu040 Advance Access Publication Date: 21 July 2014 Short Communication

Short Communication

Elizabeth M. Brown1,2,3 , Lisa R. McTaggart1,∗ , Donald E. Low2,3 and Susan E. Richardson1,3,4 1

Public Health Laboratories Toronto, Public Health Ontario, Canada, 2 Department of Microbiology, Mount Sinai Hospital, Toronto, Ontario, Canada, 3 Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada and 4 Division of Microbiology, Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada

*To whom correspondence should be addressed. Lisa R. McTaggart, Public Health Ontario, Public Health Laboratories, 81 Resources Road, Toronto, Ontario, Canada, M9P 3T1. Tel: 416-235-6543; Fax: 416-235-6281; E-mail: [email protected] Received 13 February 2014; Accepted 28 April 2014

Abstract Manipulation of Blastomyces dermatitidis requires the use of containment level 3 (CL3) practices. However, access to CL3 laboratories is limited and working conditions are restrictive. We describe the validation of a “heat-killing” method to inactivate B. dermatitidis, thus allowing cellular material to be removed from the CL3 laboratory for subsequent DNA isolation that is suitable for genetic applications. Key words: Blastomyces dermatitidis, inactivation of Blastomyces dermatitidis, deactivation of Blastomyces dermatitidis, heat inactivation.

Introduction Blastomyces dermatitidis is a dimorphic fungal pathogen responsible for causing the rare but often fatal systemic infection blastomycosis. As a risk group 3 pathogen in Canada, Australia, New Zealand, Singapore, Belgium, Germany, and the United Kingdom, the safe handling and manipulation of this fungus requires the use of containment level 3 (CL3) physical and operational practices [1–4]. In the United States, biosafety level 2 (BSL-2) and advanced BSL-2 practices are recommended for work with clinical materials, infected animals, and yeast-phase cultures, while studies involving sporulating mould cultures and environmental materials likely containing conidia necessitate biosafety level 3 practices [5]. Albeit rare, instances of laboratoryacquired infections have been reported following accidental inoculation or presumed inhalation of conidia produced by 766

mould-phase cultures [6–8]. CL3 requirements potentially hamper research on this important pathogen, since access to suitable laboratories is limited and the working environment within the CL3 laboratory can be restrictive. With clinical microbiological laboratories relying increasingly on molecular methodologies for the rapid diagnosis and genotyping of pathogenic fungi, an effective method for the inactivation of B. dermatitidis cellular material would greatly expand the opportunities of working with this organism. Confirming that the organism is noninfectious after inactivation steps would permit the removal of cellular material from the CL3 laboratory to a centralized molecular CL2 facility for the remainder of the DNA extraction procedure and genetic manipulation. Despite a few published reports of theoretical “heat-killing” steps [9–10], no study has conclusively demonstrated that B. dermatitidis is noninfectious

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Effective method for the heat inactivation of Blastomyces dermatitidis

Brown et al.

after these treatments. Our objective was to validate an effective method for the inactivation of B. dermatitidis, thus permitting its removal from the CL3 environment, while maintaining DNA integrity for subsequent molecular applications.

Materials and methods

inactivation method, one heaping loopful (10 µl) of yeast cells was transferred from a D medium agar slant culture [13] to a 2.0-ml tube containing 400 µl of Norgen resuspension buffer (Norgen Biotek, Thorold, Ontario, Canada). Suspensions were vortexed for 3 s. The inoculum was then heated and mixed by inversion at 15-min intervals at the temperature and for the duration outlined in Figure 1. Suspensions were placed in a heat block for 15 min and then in an oven for the remaining duration of the inactivation. For chemical inactivation (BBL MycoPrep), one heaping loopful (10 µl) of yeast cells was transferred to a 2.0-ml tube containing 300 µl of distilled water. The inactivation was performed according to the manufacturer’s instructions, with the volumes adjusted as follows: 300 µl of NALC–NaOH solution, 300 µl of specimen (ie, yeast-phase colonies suspended in water), and 900 µl of BBL MycoPrep phosphate buffer (pH 6.8). The suspension was incubated at room temperature for 15 min. Finally, cells were pelleted and then resuspended in 400 µl of BBL MycoPrep phosphate buffer by vortexing. To establish that the organism had been inactivated, 100 µl of either heat or chemically “inactivated” suspension was transferred to a PDA agar slant, incubated at 25◦ C for mould-phase growth, and then to a D medium agar slant incubated at 37◦ C for yeast-phase growth. At 3 weeks, inactivated material from primary agar slants was subcultured onto fresh media. Viability checks were performed on a weekly basis for a 12-week period, with inoculated primary and secondary media visually inspected for the presence or absence of growth. DNA was extracted from the remaining 200-µl suspension using the fungi/yeast genomic DNA isolation kit (Norgen Biotek) according to the manufacturer’s protocol. To confirm the integrity of inactivated DNA for downstream molecular methodologies, the internal transcribed spacer region of rDNA (its-2) and the histidine kinase gene (drk1) were amplified by polymerase chain reaction and bidirectionally sequenced as described previously [14]. DNA was quantified using the Quant-it PicoGreen dsDNA kit (Life Technologies Corp., Carlsbad, CA, USA). Identical experiments were repeated on the mould phase of B. dermatitidis using the inactivation method found to be effective at killing the yeast phase (ie, 85◦ C for 1 h).

Results

Figure 1. Study scheme for the inactivation of Blastomyces dermatitidis culture material.

Of the 23 isolates assessed for viability, 4 isolates treated with NALC–NaOH, 2 isolates exposed to 100◦ C for 20 min, and 2 isolates subjected to 60◦ C for 1 h grew during the 12 weeks following treatment (Table 1). Of these eight isolates, suspicion of breakthrough growth was observed on either the PDA or D medium agar slants by week 3, with

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The following inactivation methods were evaluated using either heat or chemical pretreatment: 100◦ C for 20 min; 60◦ C for 1 h (as previously described [9–10]); 85◦ C for 1 h; and N-acetyl-L-cysteine–2% sodium hydroxide (NALC–NaOH) solution using the BBL MycoPrep specimen digestion/decontamination kit (Becton Dickinson, Mississauga, Ontario, Canada; Fig. 1). NALC–NaOH is widely used to inhibit bacterial and fungal growth in respiratory samples prior to mycobacterial culture [11–12]. All work was performed in a biological safety cabinet within a CL3 laboratory. Twenty-three isolates of B. dermatitidis and one negative control (resuspension buffer or distilled water) were assessed for each method. A positive growth control (yeast-phase cells that were not inactivated plus resuspension buffer or water) was plated onto potato dextrose agar (PDA; Becton Dickinson) and D medium slants [13] and incubated at 25◦ C and 37◦ C, respectively. Mouldphase B. dermatitidis isolates were grown on PDA at 25◦ C for 3–7 d. Conversion to the yeast phase was achieved by subculturing mould-phase isolates onto D medium agar [13] and incubating at 37◦ C for 2–3 weeks. For each heat

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Table 1. Inactivation of Blastomyces dermatitidis and quality of DNA subsequently isolated. Inactivation procedure

1 2 3 4

100 C × 20 min 60◦ C × 1 h 85◦ C × 1 h (yeast) 85◦ C × 1 h (mould) N-acetyl-Lcysteine-2% sodium hydroxide

23 23 23 23 23

Number (%) of isolates exhibiting growth D medium slant at 37◦ C

Potato dextrose agar slant at 25◦ C

Primary

Secondary∗

Primary

Secondary∗

2 (8.7) 2 (8.7) 0 (0) 0 (0) 4 (17.4)

2 (8.7) 1 (4.3) 0 (0) 0 (0) 2 (8.7)

1 (4.3) 2 (8.7) 0 (0) 0 (0) 2 (8.7)

0 (0) 2 (8.7) 0 (0) 0 (0) 0 (0)

Total

2 (8.7) 2 (8.7) 0 (0) 0 (0) 4 (17.4)

Number (%) of isolates with internal transcribed spacer region of rDNA amplification

Number (%) of isolates with histidine kinase gene amplification

23 (100) 23 (100) 23 (100) 23 (100) 23 (100)

21 (91.3) 22 (95.7) 23 (100) 23 (100) 23 (100)

All isolates positive for growth on secondary culture were also positive for growth on the primary culture.

growth becoming more apparent by weeks 4 and 5, indicating crippling of growth, though not killing. Of the four inactivation conditions evaluated, only organisms inactivated at 85◦ C for 1 h were consistently rendered nonviable on culture and subculture over the 12-week study period. This result was replicated for mould-phase organisms inactivated at 85◦ C for 1 h (Table 1). Isolates inactivated using the other conditions demonstrated growth on either the primary agar slant and/or upon subculture to a secondary agar slant (Table 1). The chemical protocol exhibited the highest levels of breakthrough growth, with 17.4% (n = 4) of isolates demonstrating growth on either the primary or secondary agar slant. Of particular note, treatment at 60◦ C for 1h, which had previously been reported to “heat kill” B. dermatitidis [9–10], failed to inactivate all of the isolates. DNA was isolated from all strains, with the quantity obtained varying among different inactivation conditions. Heating B. dermatitidis cells at 60◦ C and 85◦ C for 1 h yielded a slightly greater concentration of DNA (0.021 ng/µl and 0.024 ng/µl, respectively) than chemical inactivation or heating at 100◦ C for 20 min (0.010 ng/µl and 0.011 ng/µl, respectively). DNA quality and suitability for downstream molecular application were assessed by amplification and sequencing of the its-2 region of the multicopy rDNA gene and the single-copy drk1 gene. The its-2 region was successfully amplified and sequenced from all isolates pretreated with any of the four methods. The drk1 gene was amplified and sequenced from all isolates except for two isolates pretreated at 100◦ C for 20 min and one isolate pretreated at 60◦ C for 1 h (Table 1).

Discussion Our results suggest that a heat pretreatment of 85◦ C for 1 h is an effective method for the inactivation of B. dermatitidis with DNA integrity suitable for molecular applications. Our preliminary assessment of a previously published method [9–10] shows that inactivation at 60◦ C

for 1 h does not consistently kill B. dermatitidis. These findings emphasize the importance of having individual laboratories validate inactivation methods prior to implementing such protocols in their laboratory.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.

References 1. American Biological Safety Association. 2013. Risk Group Classifications for Infectious Agents. Available from: http:// www.absa.org/riskgroups/fungisearch.php?genus=Blastomyces &species. Accessed 16 January 2013. 2. Human pathogens and toxins act. S.C. 2009, c. 24, Second Session, Fortieth Parliament, 57–58 Elizabeth II, 2009. 3. Public Health Agency of Canada. 2010. Pathogen data safety sheet—infectious substances: Blastomyces dermatitidis. Available from: http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss /blastomyces-dermatitidis-eng.php10. Accessed 16 January 2013. 4. Public Health Agency of Canada. Chapter 2, Biological safety, and Chapter 3, Handling infectious substances. In: Best M, Graham ML, Leitner R, Ouellette M, Ugwu K, eds. Laboratory Biosafety Guidelines, 3rd edn. Ottawa, ON: Public Health Agency of Canada, 2004: 4–26. 5. US Department of Health and Human Services. Blastomyces dermatitidis. In: Chosewood LC, Wilson DE, eds. Biosafety in Microbiological and Biomedical Laboratories, 5th edn. Washington, DC: US Department of Health and Human Services, 2009: 170–171. 6. Baum GL, Lerner PI. Primary pulmonary blastomycosis: a laboratory acquired infection. Ann Intern Med 1970; 73: 263– 265. 7. Larsh HW, Schwarz J. Accidental inoculation blastomycosis. Cutis 1977; 19: 334–336. 8. Schwarz J. Laboratory infections with fungi. In: DiSalvo AF, ed. Occupational Mycoses. Philadelphia: Lea and Febiger, 1983: 215–227.

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Number of isolates tested

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12. Reep BR, Kaplan W. The effect of newer tubercle bacillus digestion and decontamination procedures on fungi causing pulmonary diseases. Mycopathologia 1972; 46: 325– 334. 13. Kane J. Conversion of Blastomyces dermatitidis to the yeast form at 37◦ C and 26◦ C. J Clin Microbiol 1984; 20: 594– 596. 14. Brown EM, McTaggart LR, Zhang SX et al. Phylogenetic analysis reveals a cryptic species Blastomyces gilchristii, sp. nov. within the human pathogenic fungus Blastomyces dermatitidis. PLoS ONE 2013; 8: e59237.

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Effective method for the heat inactivation of Blastomyces dermatitidis.

Manipulation of Blastomyces dermatitidis requires the use of containment level 3 (CL3) practices. However, access to CL3 laboratories is limited and w...
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