Validated Methods for Removing Select Agent Samples from Biosafety Level 3 Laboratories

The Federal Select Agent Program dictates that all research entities in the United States must rigorously assess laboratory protocols to sterilize samples being removed from containment areas. We validated procedures using sterile filtration and methanol to remove the following select agents: Francisella tularensis, Burkholderia pseudomallei, B. mallei, Yersinia pestis, and Bacillus anthracis. We validated methanol treatment for B. pseudomallei. These validations reaffirm safety protocols that enable researchers to keep samples sufficiently intact when samples are transferred between laboratories.

The Federal Select Agent Program dictates that all research entities in the United States must rigorously assess laboratory protocols to sterilize samples being removed from containment areas. We validated procedures using sterile filtration and methanol to remove the following select agents: Francisella tularensis, Burkholderia pseudomallei, B. mallei, Yersinia pestis, and Bacillus anthracis. We validated methanol treatment for B. pseudomallei. These validations reaffirm safety protocols that enable researchers to keep samples sufficiently intact when samples are transferred between laboratories.
Measuring the intracellular levels of antimicrobial drugs in the BAL fluid is sometimes necessary to determine the amount of compound penetrating the site of infection within the cell. When it is necessary to measure the intracellular concentration, we treat the BAL cell pellet and the BAL fluid as 2 independent samples. Because the cell pellet sample cannot be filtered, we describe an additional procedure for removing BAL cell pellets from the containment laboratory.

Biosafety
We tested all protocols in a BSL-3 laboratory at the University of Florida, which is registered and licensed with the Centers for Disease Control and Prevention and the Animal and Plant Health Inspection Service, US Department of Agriculture, to conduct select agent research. The containment laboratory uses a high-efficiency particulate air filter to decontaminate discharged air. All staff must don facility-dedicated scrubs, Tyvek suits (Dupont, https://www.dupont. com), respiratory protection, double gloves, and shoe covers. All bacterial work is performed in a class II Biosafety cabinet, and all waste is removed using pass-through autoclaves.

Bacterial Strains and Growth Conditions
We used the following strains from the Biodefense and Emerging Infections Resource Repository: B. anthracis (Ames), Y. pestis (CO92), F. tularensis (SchuS4), B. pseudomallei (1026b), and B. mallei (China 7). We isolated B. anthracis spores according to Leighton and Doi (4) and maintained the spores in refrigerated sterile water at ≈1 × 10 10 CFU/mL. We verified this concentration by serial dilution in sterile water onto sheep blood agar plates as previously stated (5).
We cultured Y. pestis CO92 from frozen stock on sheep blood agar (Becton Dickinson, https://www. bd.com) and incubated it for 48 h at 28°C. We then removed colonies from the stock plate and suspended them in 1 mL heart infusion broth (Becton Dickinson). We added this suspension to 100 mL heart infusion broth containing 2 mL 10% xylose (Indofine, https:// indofinechemical.com). We incubated this mixture in a 500 mL flask with agitation for 18-24 h.
We then cultured B. mallei China 7 and B. pseudomallei 1026b from frozen stock vials on tryptic soy agar and incubated them at 35°C for 24-48 h to generate a stock plate of each strain. We selected 2-3 colonies from each incubated stock plate and inoculated them in brain heart infusion (BHI) broth (Becton Dickinson) overnight culture. We then incubated the cultures at 35°C with agitation for 16-20 h.
We also cultured F. tularensis SchuS4 from frozen stock onto chocolate agar (Becton Dickinson) and incubated it at 35°C for 48 h. We selected colonies from the agar plate and used them to inoculate a BHI culture containing 2% Isovitalex (Becton Dickinson). We incubated this culture for 18-20 h at 35°C with agitation.

Matrices
We tested the filtration protocol with murine lung BAL fluid, serum, plasma, and the listed culture mediums ( Table 2). For the spore preparation, we used BHI as the culture media. We purchased the murine serum, plasma, and BAL from BioreclamationIVT (https:// bioivt.com). We used mouse plasma from Balb/c mice collected in sodium citrate-containing tubes and pooled across sex. We also used mouse BAL and serum from Balb/c mice and pooled across sex.
We prepared test samples for Y. pestis from the incubated 100 mL broth culture. We took an OD reading from serially diluted broth culture and conversion factors to determine the culture concentration. We centrifuged 20 mL of this culture at 3,500 × g for 15 min. We then resuspended this pellet in 2 mL of BAL fluid ( Table 2). We repeated the process for the culture medium. We inoculated serum and plasma samples with a uncentrifuged overnight culture ( Table 2).
We prepared B. mallei test samples from the overnight broth cultures incubated previously. We prepared BAL fluid test samples by centrifuging 2 mL overnight broth culture at 3,500 rpm for 15 min and then resuspending the pellet in 2 mL BAL fluid. We inoculated serum and plasma with an overnight culture that had been diluted 1:100, then added 20 µL to each matrix ( Table 2). We inoculated culture medium by centrifuging 20 mL of the overnight culture then suspending the pellet in 2 mL of culture media ( Table 2).
We prepared B. pseudomallei test samples for culture medium as stated for B. mallei and Y. pestis using the conversion factor. We prepared BAL fluid samples by adding 200 µL overnight culture to 1.8 mL BAL fluid (Table 2). We inoculated serum and plasma with 20 µL of overnight culture that was first diluted 1:10 ( Table 2).
We prepared F. tularensis samples for culture medium with a final concentration of 2% Isovitalex. We took an OD reading and used the conversion factor to concentrate samples appropriately. We centrifuged 20 mL of an overnight culture and resuspended it in culture medium with 2% Isovitalex. We spiked serum and plasma samples with 20 µL of an overnight culture that was first diluted 1:10 and inoculated BAL fluid with 20 µL of an overnight culture (Table 2).

Methanol Test Sample Preparation
Test samples, positive controls, and the negative control of BAL fluid for the methanol treatment procedure all had a final volume of 500 µL. We used stock plates to grow bacteria, then selected colonies and suspended them in 3 mL of sterile water for injection (GE Healthcare, https://www.gehealthcare.com). We took an OD reading at 600 nm on a spectrophotometer (ThermoFisher Scientific, https://www.thermofisher.com) using a 1 cm 2 cuvette (ThermoFisher Scientific). We converted this value to an approximate CFU per milliliter value using a conversion factor as stated in test sample preparation. We calculated the total volume needed to spike each sample so that each sample would have 2 × 10 6 CFU ( Table 2).

Filtration Procedure
We conducted all filtration test procedures in triplicate for each matrix type. For negative controls, we used uninoculated matrix samples. For positive controls, we used 100 µL of unfiltered inoculated test samples suspended in broth culture medium. We then placed 450 µL of each test sample into a clean 0.2 µm PALL Nanosep Bio-Inert centrifuge filter (Pall Corporation, https://www.pall.com) with a sterile microcentrifuge tube. In accordance with the manufacturer's recommendations, we centrifuged the filters for 3 min at 14,000 × g. We then transferred the filtrate to a clean tube and sealed it to prevent secondary contamination. We emphasize that the filtrate collection tubes should not be sealed with the same cap used to close the centrifuge filter before spinning because this cap could be contaminated with residual unfiltered sample and thus might yield false positive outcomes. We then suspended the filtrate in 4.5 mL BHI and incubated it at 35°C for 2 d. We incubated the positive controls in the same manner. After 48 h, we checked the tubes for turbidity and plated 5 × 200 µL samples onto the appropriate media. We incubated these samples at 35°C for an additional 7 d to ensure complete sterility. We considered this method to be validated only if all 3 replicates of all matrices were sterile in both broth and agar medium. Any failure, defined here as positive growth on agar or in broth media, prompted a review of the procedures. Once we determined the cause of the failure, we made the appropriate adjustments and reconducted the procedure in 3 replicates.

Methanol Procedure
We centrifuged BAL fluid for 5 min at 5,000 × g. We removed the supernatant and decontaminated it using the filtration procedure detailed in the previous section. We suspended the pellet in 500 µL of 80% methanol (ThermoFisher Scientific) and incubated it for 10 min. We placed 10% of this sample into 9.5 mL Dey-Engley neutralization broth (D/E media) (Becton Dickinson) and incubated it at 35°C for 5 d. After 5 d, we plated 200 µL of the D/E media onto 5 agar plates specific to each bacterial species and incubated them at 35°C for an additional 2 d. For positive controls, we used D/E media inoculated with bacteria and D/E media with 80% methanol added to the same volume as the test sample (50 µL of 80% methanol into 9.5 mL D/E media). We incubated this tube for 10 min and then inoculated it with bacteria. We also used growth media specific to each bacterial species as positive controls. For negative controls, we used uninoculated D/E media and D/E media inoculated with methanol treated bacteria.

Results
After following the described procedures, we observed that all samples (except 1) were sterilized in broth culture after 48 h incubation. The samples remained sterile after plating on agar medium incubated for 7 d (Table 3). We determined that the test sample that had not been sterilized had sustained secondary contamination from the centrifuge filter unit cap. The PALL centrifuge filters are supplied as a filter and tube unit; they do not come with sterile secondary caps. To avoid secondary contamination, we transferred the filtrate to a clean tube immediately after spinning. We also observed that all samples were sterilized after treatment with 80% methanol and after incubation in broth culture for 5 d. The samples remained sterile on agar after an additional 2 d incubation.

Discussion
Validating sterility procedures is a time-intensive and costly necessity for removing select agent samples from BSL-3 laboratories. Researchers can streamline this process by publishing validated methods in peerreviewed journals.
We described and validated reproducible procedures for select agent sample removal. However, researchers should ascertain that none of their sample is lost because of binding to the filter material. In this study, we checked 100% of the sample as a proof of concept, although we recognize the impossibility of incubating 100% of the sample to ensure sterility during actual experiments. Our laboratory now samples 10% of the filtrate to verify successful disinfection. We have found that these filters have an approximate failure rate of 0.1%; however, other researchers such as Dauphin et al. have found a failure rate closer to 3% (14). The differences in failure rates, variety of available filter membranes, and new methods of sterilization showcase the need for clear, detailed, and reproducible published methods.

About the Author
Ms. Kesterson is a doctoral candidate in biomedical sciences at the University of Florida. Her research interests include bacterial host pathogen interaction and antimicrobial countermeasures for biothreat pathogens and their associated immune responses. The COVID-19 pandemic has caused us to reevaluate what "work" should look like. Across the world, people have converted closets to offices, kitchen tables to desks, and curtains to videoconference backgrounds. Many employees cannot help but wonder if these changes will become a new normal.
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