Heat Inactivation of Coccidioides posadasii and Coccidioides immitis for Use in Lower Biosafety Containment

Abstract

Introduction:

The difficulty involved in obtaining sufficient intact genomic deoxyribonucleic acid (DNA) from Coccidioides spp for downstream applications using published protocols prompted the exploration of inactivating mycelia and arthroconidia using heat under biosafety level 3 containment. This was followed by optimizing DNA extraction from mycelia using various methods at lower containment.

Methods:

Various exposure times and temperatures were examined to identify an effective heat inactivation procedure for arthroconidia and mycelia from both C immitis and C posadasii. Heat inactivation of mycelia was followed by DNA extraction using 2 commercially available kits, as well as a phenol:chloroform-based extraction procedure to determine DNA integrity and quantity among extraction methods using both live and heat-inactivated mycelia.

Results:

Ten-minute and 30-minute exposure times at 80°C were sufficient to inactivate Coccidioides spp arthroconidia and mycelia, respectively. DNA yield between live versus heat-inactivated mycelia was similar for each extraction procedure. However, DNA obtained using phenol:chloroform was of higher quantity and integrity compared with DNA obtained using the commercially available kits, which was highly fragmented.

Conclusion:

The ability to heat-inactivate Coccidioides cultures for processing at a lower level of containment greatly increased the efficiency of DNA extractions. Therefore, this is an ideal method for obtaining Coccidioides spp DNA and inactivated arthroconidia.

The Coccidioides genus is composed of 2 closely related species, C immitis and C posadasii, which are the etiologic agents of coccidioidomycosis, a fungal disease commonly known as Valley fever that affects humans, dogs, and other mammals. These fungal species are endemic to arid regions of North America and South America, where they exist in the soil as filamentous mycelia that alternatively segment into asexual spores known as arthroconidia.^{1 -5} Inhalation of arthroconidia is the most common route of exposure with subsequent infections ranging in severity from asymptomatic to acute or chronic pneumonia, which occasionally disseminates and can be fatal. A precise infection rate is difficult to determine due to the number of asymptomatic or otherwise undiagnosed or unreported cases, but it is estimated that the disease affects about 150 000 people per year.^6,7 These cases represent a significant disease burden in endemic regions with some symptomatic patients requiring hospitalization. In rare cases, dissemination may require lifelong antifungal treatment.^{8 -10}

Study of Coccidioides spp requires biosafety level 3 (BSL-3) containment because of the aerosol risk posed by the large amounts of arthroconidia that may be produced in standard cultures, the severity of the disease, and the lack of vaccines or other treatment options in the event of an exposure.¹¹ Therefore, BSL-3 containment is required for culture growth and extraction of nucleic acids that can subsequently be used in genetic studies.^{12 -16} A variety of molecular methods are being used in these genetic studies, including polymerase chain reaction, microsatellite analysis, recombinant deoxyribonucleic acid (DNA) amplification, and whole-genome sequencing. Each of these methods produces the best results with high-quality and quantity intact genomic DNA (gDNA). There are many commercially available DNA extraction kits. However, fungal cell walls can be resistant to the standard lysis methods used in these kits, which may result in low yields and/or fragmented DNA. Higher quality and quantity DNA may be obtained using phenol:chloroform based extractions.^{17 -19} Unfortunately, this method produces a mixture of infectious agent and chemical hazardous waste. Such waste is burdensome to manage and remove from containment, as the chemicals must either be validated as effective at inactivating the agent or the waste must be treated to inactivate the potentially infectious component before it can be removed from containment for disposal as chemical waste.^20,21 In addition, these extraction methods are time intensive, potentially requiring multiple days of work inside BSL-3 containment.

The best way to simplify this process is to inactivate agent samples, remove the inactivated samples from BSL-3 containment, and then complete nucleic acid extractions at a lower level of containment. Previously, autoclaving was used to inactivate cultures of C immitis, which were then lyophilized and extracted using phenol:chloroform in a lower containment setting. This method can yield adequate amounts of DNA for polymerase chain reaction. However, the resultant DNA included a spectrum of fragment sizes, and broad differences in DNA quantity were found among strains.²² Thus, we developed an alternative method that could produce a greater quantity of DNA with larger fragments of consistent size for whole-genome sequencing applications. Therefore, a heat inactivation method for Coccidioides spp arthroconidia and mycelia was developed, and the relative quantity and integrity of DNA produced was assessed.

Materials and Methods

Heat inactivation was performed on both mycelia and arthroconidia from C posadasii strain C735 (American Type Culture Collection 96335) and C immitis strain RS. Liquid cultures of mycelia were grown in 50-mL vented centrifuge tubes (BD-Falcon VWR) in 30 mL 2× GYE (2% w/v glucose; [VWR] and 1% w/v Difco yeast extract [BD Biosciences]). Cultures were inoculated with either a 1 × 1 cm agar plug of existing fungal growth or 50 to 100 μL existing arthroconidia suspended in 1× phosphate-buffered saline (PBS) (Gibco) and then incubated for a minimum of 72 hours at 30°C with shaking at 250 rpm. The liquid mycelia cultures were then filtered using 100-μm nested filters (Corning, Fisher Scientific) and rinsed with 1× PBS. For inactivation tests, 2 heaping 10-μL loopfuls of mycelia were suspended in 500 μL 1× PBS in a 1.5-mL microcentrifuge tube (Thumbs-up, Diversified Biotech). Arthroconidia for each strain were harvested as previously described.²³ Arthroconidia suspensions were quantified by fixing an aliquot of each suspension in 1% formalin for 48 hours and performing counts using a hemocytometer. For inactivation tests, arthroconidia from both strains were adjusted to 1.4 × 10⁵ arthroconidia/mL using 1× PBS, and aliquots of 500 μL were dispensed into a 1.5-mL microcentrifuge tube.

The mycelia and arthroconidia suspensions were used to determine an effective temperature and exposure time for heat inactivation of Coccidioides spp material. Both a temperature curve and an exposure time curve were performed with 3 replicates for each treatment. Exposure times of <1, 2, 5, 10, 20, and 30 minutes were tested by incubating the mycelia and arthroconidia suspensions in an 80°C heat block for the tested time and then immediately plating 100% of each sample on 2× GYE. Exposure temperatures of 65°C, 70°C, and 75°C were tested using a fixed incubation time of 10 minutes for arthroconidia and 30 minutes for mycelia, followed by immediate plating of 100% of each sample on 2× GYE.

In an effort to increase DNA yield, additional inactivation tests were performed using mycelia suspended in fungal lysis buffer (60% molecular grade water [Invitrogen], 10% v/v 3 mol/L NaOAc at pH 6.0 [Sigma-Aldrich], 5% w/v 10% sodium dodecyl sulfate [VWR International], 20% v/v 0.5 mol/L ethylenediaminetetraacetic acid at pH 8.0 [Amresco, VWR International], 5% v/v 1 mol/l Tris-HCl at pH 7.5 [Fisher Scientific], and 0.1% 2-mercaptoethanol [Sigma Aldrich]). For these tests, 2 heaping 10-μl loopfuls of mycelia were suspended in 1-mL fungal lysis buffer in a 1.5-mL microcentrifuge tube. Exposure times of 20 and 30 minutes were tested by incubating 3 replicates at each time point in an 80°C heat block and then immediately plating 100% of each sample on 2× GYE.

Process controls for each resuspension buffer were also analyzed. These process controls consisted of heating a single replicate of both uninoculated 1× PBS (500 μL) and fungal lysis buffer (1 mL) at each time and temperature point as the live samples. Following incubation, these replicates were inoculated with live fungal culture and plated to determine if the heated resuspension buffers would affect growth of viable fungal cells. Two additional process controls were analyzed for the fungal lysis buffer to account for potential growth inhibitory effects of the lysis buffer. The first of these consisted of fungal lysis buffer heated and inoculated as above, followed by filtration of the lysis buffer, rinsing with 1× PBS, and plating of the mycelia pellet. For the second of these, fungal lysis buffer was heated as above, removed from the 1.5-mL microcentrifuge tube, an equal amount of 1× PBS added to the microcentrifuge tube, the PBS inoculated with live fungal culture, and the inoculated PBS plated. All test plates were incubated at 30°C for 14 days and examined for any evidence of growth.

Fungal DNA was extracted using equivalent amounts of live and heat-inactivated mycelia. Fungal samples suspended in 1× PBS were placed in 2-mL screw-cap tubes containing 1.0 mm silica acid washed glass beads 2.5 g/mL (Sigma-Aldrich) and 1 mL lysis buffer. Fungal samples suspended in lysis buffer were added directly to the 2 mL screw-cap tubes containing the glass beads. These tubes were bead-beaten on a vortex adaptor for 10 minutes at maximum speed. Samples were then incubated in a heat block at 65°C for 30 minutes followed by centrifugation at 10 000g for 2 minutes. Approximately 650 μL supernatant was transferred to a 1.5-mL microcentrifuge tube containing an equal volume of phenol:chloroform:isoamyl 25:24:1 (Sigma-Aldrich) and mixed by inverting the tubes for 10 minutes. These tubes were centrifuged at 14 000g for 10 minutes and the aqueous layer (∼600 μL) transferred to a fresh 1.5-mL microcentrifuge tube containing an equal amount of chloroform:isoamyl (24:1), which was mixed by inverting the tubes for 2 minutes. These tubes were centrifuged at 20 000g for 10 minutes at 4°C, the supernatant (∼500 μL) was transferred to a third 1.5-mL microcentrifuge tube containing 2/3 the volume of the transferred supernatant of ice-cold isopropanol and incubated at –20°C overnight. The following day, the tubes were centrifuged at 20 000g for 10 minutes at 4°C to pellet the DNA, the isopropanol decanted, and 500 μL cold 70% ethanol added to wash the pellet. The DNA was again pelleted by centrifuging at 20 000g for 5 minutes, the ethanol decanted, and 300 μL cold 95% ethanol added for a second wash step. The purified DNA was pelleted by centrifuging at 20 000g for 5 minutes, decanting the ethanol, and allowing residual ethanol to evaporate by placing the open tubes at an angle. The purified DNA was resuspended in 50 μL 1× TE (10 mmol/L Tris-HCl and 1 mmol/L ethylenediaminetetraacetic acid; Fisher Scientific) and incubated at 4°C overnight prior to gel quantification.

For comparison of kit-obtained DNA yield, DNA was also extracted from both live and heat-inactivated mycelia using 2 commercially available kits, Microbial DNA Isolation Kit (currently known as DNeasy UltraClean Microbial Isolation Kit, Qiagen) and UltraClean Tissue and Cells DNA Isolation Kit (MoBio). Extractions were performed according to manufacturer’s direction using 2 heaping 10 μL loopfuls of mycelia from each strain.

Gel electrophoresis for 1.75 hours at 95 V on 0.7% agarose (Fisher) with 0.1% Sybr Safe (Invitrogen) was used to visualize and quantify the DNA. HindIII Lambda Ladder (New England BioLabs) with blue loading dye (New England BioLabs) was used for comparative analysis. This was prepared using 2 μL heat-denatured ladder, which corresponds to 1 μg DNA. Each quantified sample consisted of 5 μL of the prepared fungal DNA, 5 μL 1× TE, and 2 μL loading dye. For each DNA extraction method, we identified the lowest and highest intensity band and followed the calculation from New England BioLabs to determine concentration.²⁴ The number of base pairs for each fragment of interest was divided by the number of base pairs of the total uncut ladder molecule (48 502 bp) and multiplied by the total input (1 μg). Using this calculation, 23 130 bp is equivalent to 477 ng and 564 bp is equivalent to 12 ng. To determine overall yield, the concentration of the fragment was divided by the DNA input volume (5 μL) and multiplied by the total volume of extracted DNA (50 μL).

Results

Both Coccidioides spp arthroconidia and mycelia were effectively inactivated using heat. Arthroconidia were inactivated at 80°C after 5 minutes of exposure for both species. Mycelia from C posadasii strain C735 were also inactivated after 5 minutes of exposure. However, C immitis strain RS mycelia exhibited growth on one replicate after 10 minutes of exposure (Table 1). Thus, we increased the exposure times to 10 and 30 minutes for use in the final heat inactivation procedure for arthroconidia and mycelia, respectively, to incorporate an additional margin of safety. The increased contact times resulted in complete inactivation of both arthroconidia and mycelia at all of the tested temperatures (65°C, 70°C, and 75°C) (Table 2). A temperature of 80°C was chosen for both Coccidioides spp arthroconidia and mycelia to further increase the margin of safety in the final procedure. We have observed that different brands of tubes can affect heat killing, and we recommend additional testing if using alternative tubes (especially larger 2-mL or skirted tubes). As expected, fungal growth was observed in all process controls in which live arthroconidia or mycelia were inoculated into heat-treated 1× PBS, indicating that heating PBS does not inhibit growth. Fungal growth was not observed when mycelia were inoculated into heat-treated fungal lysis buffer and plated without a PBS wash step, likely because of the inherent inactivation properties of the lysis buffer.

Table 1.

Heat Inactivation of Coccidioides spp at 80°C at Varying Exposure Times.

	Exposure Time (min), 80°C
Agent	<1	2	5	10	20	30
C735 mycelia	+ + +	+ – –	– – –	– – –	– – –	– – –
C735 arthroconidia	+ + +	+ + –	– – –	– – –	– – –	– – –
RS mycelia	+ + +	+ + –	– – –	+ – –	– – –	– – –
RS arthroconidia	+ + +	+ + +	– – –	– – –	– – –	– – –

Abbreviations: +, positive for growth; –, negative for growth.

Table 2.

Heat Inactivation of Coccidioides spp at Varying Temperatures.

	Exposure Temperature
Agenta	65°C	70°C	75°C	80°C
C735 mycelia	– – –	– – –	– –	– – –
C735 arthroconidia	– – –	– – –	– – –	– – –
RS mycelia	– – –	– – –	– – –	– – –
RS arthroconidia	– – –	– – –	– –	– – –

Abbreviation: –, negative for growth.

^a Coccidioides spp arthroconidia and mycelia were exposed to each tested temperature for 10 minutes and 30 minutes, respectively.

DNA integrity and quantity were comparable for live and heat-inactivated Coccidioides spp mycelia extracted using a phenol:chloroform protocol. All replicates yielded high–molecular weight gDNA (23 130 bp; Figures 1 and 2). Total gDNA yields for both the live and inactivated mycelia suspended in 1× PBS were similar between replicates and yielded substantial gDNA (420-900 ng; Figure 1). Relative yields were increased for live and inactivated mycelia suspended in fungal lysis buffer (0.480-4.77 μg; Figure 2). Using commercially available kits (Microbial DNA Isolation Kit and UltraClean Tissue and Cells DNA Isolation Kit), gDNA exhibited a range of fragment sizes (564-9416 bp) and lower yields (120-420 ng; Figure 3). Therefore, no meaningful differences were detected in gDNA yields between live and heat-inactivated Coccidioides spp mycelia for either of the commercially available kits.

Figure 1.

Phenol:chloroform-extracted genomic DNA from live versus heat-inactivated C posadasii strain C735 and C immitis strain RS mycelia in PBS. Mycelia were suspended in 1× PBS and heat-inactivated at 80°C for 30 minutes before being extracted.

Figure 2.

Phenol:chloroform-extracted genomic DNA from live versus heat-inactivated C posadasii strain C735 and C immitis strain RS mycelia in fungal lysis buffer. Mycelia were suspended in fungal lysis buffer and heat-inactivated at 80°C for 30 minutes before being extracted.

Figure 3.

Commercial kit–extracted genomic DNA from live versus heat-inactivated C posadasii strain C735 and C immitis strain RS mycelia in PBS. Mycelia were suspended in 1× PBS and heat-inactivated at 80°C for 30 minutes before being extracted. Two commercial kits were used, Microbial DNA Isolation Kit (MK) and UltraClean Tissue and Cells DNA Isolation Kit (UK).

Discussion

Overall, our data show that heat is effective at inactivating Coccidioides spp arthroconidia and mycelia. Arthroconidia were effectively inactivated within 10 minutes at multiple temperatures. However, mycelia required a somewhat longer exposure time, possibly because of the increased amount of biomass, but were still effectively inactivated within 30 minutes at multiple tested temperatures (Table 2). For both cell types, 80°C is recommended to increase the margin of safety for the final inactivation procedures, as arthroconidia and mycelia were effectively inactivated within 5 and 20 minutes, respectively (Table 1). These results were consistent with previous results showing inactivation of Coccidioides spp mycelia at 100°C within 15 minutes.²¹ A single replicate of C immitis strain RS mycelia exhibited growth after 10 minutes of exposure at 80°C, although all other replicates were inactivated after 5 minutes of exposure (Table 1). This could indicate strain or species variation in sensitivity to the heat inactivation procedure or could simply be the result of stochastic variation. Further inactivation experiments with additional strains of C immitis could provide more insight into this result. To date, this protocol has been used to effectively inactivate 37 additional strains of C posadasii and 25 strains of C immitis mycelia with sufficient gDNA for whole-genome sequencing without issue. We recommend verification viability testing for any sample removed from a BSL-3 laboratory.

The simple heat inactivation procedure presented here can rapidly produce inactivated fungal material for use in downstream analyses that can be performed at lower containment levels. We showed no loss in DNA integrity or quantity when comparing DNA from heat-inactivated or live mycelia. The ability to complete DNA extractions at lower containment reduces costs because of both the significant reduction in laboratory time and a reduction in necessary personal protective equipment required by protocols spanning multiple days. Importantly, disposal of the chemical waste produced during the phenol:chloroform DNA extraction is simplified at a lower containment level.

The superior DNA integrity and yield combined with the minimization of the major drawbacks to the phenol:chloroform extraction procedure (e.g., time and chemical waste disposal) afforded by the use of heat-inactivated Coccidioides spp mycelia represents a strong argument for the routine use of this procedure over other DNA extraction methods for fungal samples. The lower DNA yields provided by commercially available kits frequently requires multiple extractions be performed to obtain sufficient DNA for various applications, increasing the cost and reducing the advantages associated with the convenience of these kits. In addition, small DNA fragments prevent the use of long read sequencing applications. Performing the Coccidioides spp mycelia heat inactivation in fungal lysis buffer, rather than PBS, further simplified the process because the inactivated mycelia could be transferred directly into a bead tube following inactivation. This change allowed (1) increasing the margin of safety because of the added inactivation properties of the lysis buffer and (2) further increasing DNA integrity and quantity. Each experiment started with equivalent amounts of starting material; therefore, the increase in DNA quantity and integrity is likely due to elimination of the 1× PBS buffer, which diluted the fungal lysis buffer and also increased the total volume during the bead beating step. Using this method, we obtained higher molecular weight gDNA than in previously published work using heat inactivation. Although not explicitly tested, we suggest that gDNA integrity was maintained because of the lower exposure temperature and yield increased because of chemical properties of the lysis buffer.

Although not explored here, we anticipate that the inactivated arthroconidia produced by the heat inactivation procedure presented here will be equally useful. For example, heat-inactivated arthroconidia could be used for applications such as microscopy, phagocytic assays, or vaccine development. Interestingly, human phagocytic cells have shown taxis toward formalin-fixed cultures of Coccidioides spp, and heat inactivation could provide a chemical-free alternative.²⁵

These results build on previously published work by expanding the heat inactivation method to both species and key lifecycle stages of Coccidioides. Additionally, we derived the required temperature and exposure time to inactivate the fungi using a specific biomass rather than culture size, which can fluctuate on the basis of arthroconidia input or growth rate differences among strains. Finally, this method explores the inhibitive properties of the solution buffers (PBS or lysis buffer) to ensure that fungal growth is not inhibited because of the presence of a particular solution, increasing confidence in future viability tests of inactivated samples.

Conclusions

In summary, we present a simple and effective method for heat-inactivating Coccidioides spp arthroconidia and mycelia. The use of this procedure should, of course, be validated in-house by other laboratories planning to use it. Routine verification of a percentage of each sample or production lot is also recommended to ensure effective inactivation, as is the use of a calibrated thermometer to verify heat block temperature. Importantly, this procedure reduces the time spent handling active cultures and increases overall biosafety. Inactivated Coccidioides spp materials produced using this method can be safely transferred to lower containment and used in a wide variety of downstream procedures that may not be easily perform in BSL-3 laboratories.

Ethical Approval Statement

Authors declare no work was performed on human subjects or laboratory animals during the course of the study and was not subjected to a formal approval by a relevant ethics committee or institutional review board.

Statement of Human and Animal Rights

No human subjects or animals were used.

Statement of Informed Consent

No written or oral informed consent statements were required for this study.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Funding to support this work was provided by grants to B.M.B. from ABRC (14-082975, 16-162415) and the National Institute of Allergy and Infectious Diseases (R21AI28536).