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Inactivation of Brucella suis, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis Using Vaporous Hydrogen Peroxide James V. Rogers, William R. Richter, Morgan Q. S. Wendling, and Adrienne M. Shesky Battelle Memorial Institute, Columbus, Ohio

Abstract This study evaluated the inactivation of Brucella suis, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis on glass, Hypalon® rubber glove, and stainless steel using vaporous hydrogen peroxide fumigation of a ~15 m3 chamber. A suspension of approximately 1 x 108 colony forming units (CFU) of each organism was dried on coupons of each type of test surface and exposed to vaporous hydrogen peroxide. A significant reduction in the log10 CFU of each organism on all test materials was observed between the controls evaluated after a 1-hour drying time and unexposed controls evaluated after decontamination. For all organisms, qualitative growth assessments showed that vaporous hydrogen peroxide exposure completely inactivated bacterial viability on all replicates of the test materials incubated up to 7 days post-exposure. In parallel, all Geobacillus stearothermophilus biological indicators (BI) exposed to vaporous hydrogen peroxide exhibited no growth after 1 and 7 days incubation. This study provides information on using a combination of quantitative and qualitative growth assessments to evaluate vaporous hydrogen peroxide for the surface decontamination of B. suis, B. pseudomallei, F. tularensis, and Y. pestis within a large-scale chamber.

Introduction The biological agents Brucella suis, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis possess the capacity to cause human infection and are listed by the Centers for Disease Control and Prevention (CDC) as potential bioterrorism agents (www.bt.cdc.gov/ agent/agentlist.asp). Both F. tularensis and Y. pestis are considered Category A agents due to their potential for the greatest adverse impact on public health resulting in mass casualties and public fear (Dennis et al., 2001; Inglesby et al., 2000; Rotz et al., 2002). Category A agents also require a high level of public health preparedness and possess a moderate to high potential for large-scale dissemination (Rotz et al., 2002). The Category B agents, B. suis and B. pseudomallei, have a lower potential for large-scale release and a decreased capacity to cause illness and death, and require fewer public health preparedness efforts when compared to Category A agents (Rotz et al., 2002).

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Although bacterial spores (e.g., Bacillus anthracis) can persist in the environment for long periods of time, vegetative bacteria are susceptible to changes in environmental conditions in which the viability of these organisms can be drastically reduced. The environmental persistence of bacterial agents is an important piece of information to estimate the residual risk of transmission and infectivity. Moreover, the duration of biological agent viability with respect to the environmental matrix (e.g., water, soil, fomite) and conditions (e.g., temperature and relative humidity) can help determine the appropriate decontamination approach (e.g., fumigation) needed to treat an exposure site. Sinclair et al. (2008) provide information on the environmental persistence of biological select agents, which could be used to support the decision regarding the type and extent of decontamination needed in the event of a biological agent release and contamination event. Fumigants (e.g., gases or vapors) are advantageous for treating large-volume areas due to their ease of dispersion and extensive surface area coverage. A common approach for decontamination and disinfection is the implementation of vaporous hydrogen peroxide, which has been used to treat pharmaceutical facilities, hospital rooms, ambulances, animal holding rooms, air ducts, mail facilities, and two office buildings (Anderson et al., 2006; Canter et al., 2005; Dryden et al., 2008; French et al., 2004; Kahnert et al., 2005; Krause et al., 2001; Verce et al., 2008). Moreover, vaporous hydrogen peroxide has been shown to be effective in inactivating a wide range of microorganisms (Boyce et al., 2008; French et al., 2004; Grare et al., 2008; Hall et al., 2007; Hall et al., 2008; Heckert et al., 1997; Johnston et al., 2005; Klapes & Vesley, 1990; Otter & Budde-Niekiel, 2009; Otter & French, 2009; Rastogi et al., 2009), including biological agents (Rogers et al., 2005; Rogers et al., 2008; Rogers & Choi, 2008; Rogers et al., 2009). Aerosols are considered the most likely and effective approach to disperse biological threat agents whether they are bacteria, viruses, or toxins (Pitt & LeClaire, 2007), thus prompting the need for aerosolrelated studies in support of biological defense research operations. Our facility possesses a custom-made biosafety level 3 (BSL-3) aerosol research and component assessment (ARCA) chamber for the testing and evaluation of sensors, detectors, decontaminants, or other large-scale studies using aerosolized biological agents. This ARCA chamber has an internal working volume of

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approximately 15 m3 and a working surface area of approximately 59 m2 (Rogers et al., 2009). Due to its size, the ARCA chamber must be fully decontaminated to maximize safety and prevent potential secondary aerosolization and exposure. Recently, we demonstrated the inactivation of B. anthracis spores in the ARCA chamber using vaporous hydrogen peroxide (Rogers et al., 2009); however, many other agents can be used and aerosolized, so inactivation needs to be demonstrated. Therefore, the purpose of this study was to demonstrate the ability to inactivate Brucella suis, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis dried onto materials within the ARCA chamber. The results of this study can provide other end users, safety experts, and regulatory agencies with information regarding the inactivation of these biological agents using vaporous hydrogen peroxide.

Materials and Methods Test Organisms

All portions of testing were performed under BSL-3 conditions in accordance with the Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th edition. The organisms used for testing included B. suis biotype I, B. pseudomallei 1026B, F. tularensis Schu S4, and Y. pestis CO92. For testing, suspensions of each organism were prepared from a single bacterial colony inoculated into 10 mL liquid cultures that were agitated on an orbital shaker set to 200 rpm. Specifically, B. suis was grown in brain heart infusion (BHI) broth (BD Diagnostic Systems, Sparks, MD), and B. pseudomallei was cultured in tryptic soy broth (TSB) (Remel, Lexena, KS); both organisms were cultured at 37ºC. The F. tularensis was grown at 37ºC in Mueller-Hinton broth supplemented with 0.025% iron pyrophosphate, 0.1% glucose, and 2% IsoVitaleX™ (BD Diagnostic Systems), while the Y. pestis was grown in TSB at 26ºC.

Test Materials

Three materials commonly found in the ARCA chamber (The Baker Company, Sanford, ME) were used as the test surfaces; these included glass, Hypalon® glove (The Baker Company), and stainless steel (Adept Products, Inc., West Jefferson, OH). The glass coupons were microscope slides (Fisher HealthCare, Houston, TX), the Hypalon® coupons (approximately 2.0 cm x 2.0 cm) were cut from a Hypalon® glove, and the stainless steel coupons were approximately 1.5 cm x 6.5 cm. All coupons were autoclaved at 121ºC for 20 minutes prior to testing, and a visual assessment of each coupon was performed to ensure that no physical changes or defects occurred due to the sterilization process.

Decontamination Procedure

For each organism, each type of test coupon was inoculated in a Biological Safety Cabinet (BSC) Class III 26

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with approximately 1.0 x 108 CFU by dispensing ten, 10 µL droplets across each coupon surface as previously described (Rogers et al., 2009). The droplets were allowed to dry for 1 hour prior to decontamination. For each test material and organism combination, three inoculated coupons and one blank (not inoculated) were used for decontamination. In parallel, two sets of inoculated coupons (N=3/material) and blanks (N=1/material) were used as controls. The first set was used to control for any potential decrease in microorganism viability during the 1-hour drying time. The second set of control coupons were maintained in a separate, isolated BSC III to control for any potential decrease in microorganism viability during the entire decontamination run. Following the 1-hour dry time, the inoculated coupons intended for decontamination (and one blank) were transferred into the ARCA chamber. One of each coupon type was placed in the upstream, middle, and downstream sections to demonstrate decontamination efficacy throughout the ARCA chamber. Four 12-inch fans were placed inside the ARCA chamber to provide turbulence for maximizing vaporous hydrogen peroxide distribution. The 1-hour drying control coupons (and blanks) were processed for bacterial viability as described in the Sample Processing section below. The decontamination control coupons (and blanks) were transferred to a Plas Labs Model 830-ABC Compact Glove Box (Plas Labs, Inc., Lansing, MI), and the coupons were placed lying flat, inoculated surface side up. In parallel, single species biological indicators (BI) containing approximately 1 x 106 Geobacillus stearothermophilus (ATCC 12980) spores on stainless steel disks sealed in Tyvek pouches (Apex Laboratories, Inc., Apex, NC) were used to evaluate the decontamination process. Sixteen BI were placed at separate locations within the ARCA chamber and subjected to decontamination; a single BI affixed to the outside of the ARCA was not decontaminated and was used as a positive control. Additionally, VHP Chemical Indicator NB305 strips (STERIS Corporation, Mentor, OH) were used to provide a visual indication (color change) that fumigation of the ARCA chamber had occurred. Sixteen indicator strips were placed at the same locations within the ARCA as the BI, while a single indicator strip was placed with the non-decontaminated control BI. Following the decontamination run, all indicator strips were visually inspected for a color change. The decontamination procedure was performed using the VHP® 1000ED Biodecontamination System (STERIS Corporation), and identical fumigation parameters were used as previously described (Rogers et al., 2009). Briefly, the decontamination process consisted of dehumidification (10 minutes; 6.9 mg/L relative humidity), conditioning (20 minutes; 6.5 g/min hydrogen peroxide), decontamination (5.5 hours; 5.0 g/min hydrogen peroxide), and aeration phases (>4 hours), which were all controlled by the hydrogen peroxide generator. During the decontamination run, the hydrogen peroxide concen-

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tration, relative humidity, and temperature were monitored in real-time using a datapoint capture frequency of 1 minute. The hydrogen peroxide was monitored using an ATI Series B12 two-wire gas transmitter connected to a 01000 ppm remote hydrogen peroxide sensor (Analytical Technology, Inc., Collegeville, PA) suspended approximately 1 m from the middle of the ARCA chamber ceiling. The temperature and relative humidity were monitored in real-time using a HOBO U12-006 data logger (Onset Computer Corporation, Bourne, MA) that was placed in the middle of the ARCA main chamber. In the control chamber, temperature and relative humidity were monitored using a Yokogawa DX2010 (Yokogawa Electric Corporation, Tokyo) connected to an Omega HX93AC temperature/relative humidity probe (Omega Engineering Inc., Stamford, CT). Two separate decontamination runs were performed on two separate days. The first run included B. suis and B. pseudomallei, while the second run included F. tularensis and Y. pestis.

Sample Processing

To evaluate the effectiveness of vaporous hydrogen peroxide against B. suis, B. pseudomallei, F. tularensis, and Y. pestis, a combination of both quantitative and qualitative growth assessments was used as described previously (Rogers et al., 2008). The quantitative approach enabled an evaluation of decreases in organism viability as a result of the initial drying of the inoculum as well as the time period in which the non-decontaminated controls were maintained in parallel with the decontamination run. Following the 1-hour drying time, one set of control coupons (inoculated and blank coupons for each material type) for each organism was placed into 50 mL conical tubes (one coupon per tube) containing 10 mL of sterile phosphate-buffered saline (PBS) (Sigma, St. Louis, MO), and bacteria were extracted by agitating the tubes at 200 rpm on an orbital shaker for 15 minutes at room temperature. Following extraction, a 1.0 mL aliquot of

each extract was removed and ten-fold serial dilutions were prepared in sterile PBS. Aliquots (100 µL) of the undiluted extract and each serial dilution were plated in triplicate onto BHI agar (BD Diagnostic Systems) for B. suis, tryptic soy agar (Hardy Diagnostics, Santa Maria, CA) for B. pseudomallei and Y. pestis, or Chocolate II agar with hemoglobin and IsoVitaleX™ (BD Diagnostic Systems) for F. tularensis. The plates were incubated for 18-24 hours (B. pseudomallei) or 48-72 hours (B. suis, F. tularensis, and Y. pestis) at 26ºC (Y. pestis) or 37ºC (B. pseudomallei, B. suis, and F. tularensis). Following the decontamination run, the second set of control coupons and corresponding blanks for each organism were placed into 50 mL conical tubes (one coupon per tube) containing 10 mL of sterile PBS, extracted, and serial dilutions were prepared as described above. Following incubation, all plates were enumerated, average counts for each triplicate were calculated, and CFU/mL determined by multiplying the average number of colonies per plate by the reciprocal of the dilution. Data were expressed as the mean ± standard deviation (SD) of observed CFU. Following the decontamination run, each test coupon and BI were removed from the ARCA chamber, individually placed into 50 mL conical tubes containing 20 mL of the appropriate liquid culture medium, and cultured at the specific temperature for each organism. The G. stearothermophilus BI were cultured at 55º-60ºC in TSB. All cultures were visually inspected for growth (turbid culture) or no growth (clear culture) after incubating for 1 and 7 days.

Statistical Analysis

Data were expressed as mean log10 CFU of recoverable bacteria ± SD. The t-test (MS Excel; Microsoft Corporation, Redmond, WA) was used to compare the mean log10 CFU between the 1-hour drying controls and the decontamination controls. P0.05 was used as the level for significance.

Figure 1

Representative real-time profile of vaporous hydrogen peroxide concentration in the ARCA chamber.

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Results Decontamination Run

During the first run, the hydrogen peroxide concentration of the conditioning phase peaked to approximately 420 ppm and decreased to a level of approximately 230 ppm during the decontamination phase. The second run showed a similar profile with a conditioning phase peak of approximately 360 ppm that leveled off at approximately 265 ppm during the decontamination phase. Figure 1 provides a representative hydrogen peroxide concentration profile in the ARCA chamber during an entire decontamination run. The relative humidity also peaked during conditioning to approximately 100% and 98% for runs 1 and 2, respectively, and slowly decreased during the decontamination phase. The mean (±SD) temperatures in the ARCA during decontamination runs 1 and 2 were 22.0 ± 0.4ºC and 21.8 ± 0.6ºC, respectively. In the control chamber for both runs, the temperature and relative humidity ranged between 23º-24ºC and 45%-50%, respectively. Following decontamination, all chemical indicator strips present in the ARCA chamber during the decon-

tamination run changed color, indicating exposure to the vaporous hydrogen peroxide. The control chemical indicator strips did not change color. Upon visual inspection, no physical damage was observed for the hydrogen peroxide-exposed coupons.

Decontamination Efficacy

For all organisms on the three types of coupon materials, the 1-hour drying controls and unexposed decontamination controls exhibited recoverable, viable organisms (Table 1). For the 1-hour drying controls, the total number of recoverable organisms ranged from 4.7 log10 CFU for Y. pestis on glass to 8.3 log10 CFU for B. suis on glass. For the decontamination controls, the total number of recoverable organisms ranged from 3.2 log10 CFU for Y. pestis on glass to 7.6 log10 CFU for B. suis on stainless steel. For all organisms on the three types of coupon materials, the mean recoverable log10 CFU for each of the decontamination controls was significantly lower (P0.05) than the mean recoverable log10 CFU for the 1-hour drying controls (Table 1). For all coupon types, vaporous hydrogen peroxide fumigation resulted in the complete inactivation of

Table 1

Mean Total Log10 CFU of B. suis, B. pseudomallei, F. tularensis, and Y. pestis Recovered (±SD) from the 1-hour Drying and Decontamination Controls (N=3/material)

B. suis Glass

Organism/Test Material and Treatment

Hypalon® Glove Stainless Steel B. pseudomallei Glass Hypalon® Glove Stainless Steel F. tularensis Glass Hypalon® Glove Stainless Steel Y. pestis Glass Hypalon® Glove Stainless Steel

Mean Total Log10 CFU Recovered (±SD)

1-hour Drying Control Decontamination Control 1-hour Drying Control Decontamination Control 1-hour Drying Control Decontamination Control

8.3 ± 0.23 7.5 ± 0.04* 8.0 ± 0.06 7.4 ± 0.08* 8.1 ± 0.09 7.6 ± 0.09*


7.9 ± 0.03 5.5 ± 0.06* 7.8 ± 0.01 5.7 ± 0.23* 7.9 ± 0.04 5.7 ± 0.22*


5.8 ± 0.06 4.6 ± 0.11* 5.8 ± 0.14 4.6 ± 0.09* 7.7 ± 0.01 4.4 ± 0.11*


4.7 ± 0.04 3.2 ± 0.08* 6.0 ± 0.01 3.4 ± 0.05* 4.9 ± 0.13 3.4 ± 0.22*

*Mean value is significantly different than the 1-hour drying controls (P0.05).

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B. suis, B. pseudomallei, F. tularensis, and Y. pestis, as demonstrated by the lack of viable growth in liquid cultures through 7 days post-exposure (Table 2). All control blanks and decontamination blanks were negative for growth (Table 2). For all samples and organisms tested, the bacterial colonies observed were a homogenous mixture and the identification of these colonies was confirmed by comparing the colony morphology from the coupon sample extracts to that of the respective stock suspension grown on plates. For all BI exposed to the vaporous hydrogen peroxide, no growth was observed as determined by the lack of visibly cloudy liquid cultures through 7 days incubation post-exposure (Table 2). All of the unexposed BI exhibited growth as determined by the presence of visibly cloudy liquid cultures (Table 2).

Discussion This study demonstrates that virulent B. suis, B. pseudomallei, F. tularensis, and Y. pestis were inactivated when inoculated on three material surfaces and exposed to vaporous hydrogen peroxide within a large aerosol-generating chamber. In parallel, BI containing G. stearothermophilus spores exposed to vaporous hydrogen peroxide were completely inactivated as demonstrated by the lack of growth (no turbidity) in the liquid cultures after 7 days of incubation, providing a qualitative assessment for evaluating decontamination performance as previously indicated (French et al., 2004; Heckert et al., 1997; Hillman, 2004; Johnston et al., 2005; Rogers et al., 2005; Rogers & Choi, 2008; Rogers

et al., 2008; Rogers et al., 2009; Sigwarth & Stark, 2003). Previously, both F. tularensis and Y. pestis dried onto non-porous surfaces were completely inactivated by vaporous hydrogen peroxide in chambers ranging in volume up to approximately 3,600 liters (Rogers & Choi, 2008; Rogers et al., 2008). Brucella suis and B. pseudomallei have been shown to be inactivated by freechlorine, monochloramine and ultraviolet light (O’Connell et al., 2009; Rose et al., 2007; Sagripanti et al., 2009); however, this study provides the first demonstration of inactivation by using vaporous hydrogen peroxide, especially on a large-scale. To make informed decisions regarding the potential health risks associated with microorganisms that are known to be infectious at low levels, generating experimental data that demonstrate complete kill on material surfaces is pertinent. B. suis, B. pseudomallei, F. tularensis, and Y. pestis can persist outside a host for extended periods of time (Bossi et al., 2004; Inglis & Sagripanti, 2006; Sinclair et al., 2008), and their respective estimated infectious doses can range from less than 10 to thousands of organisms (Bossi et al., 2004; Hoppe et al., 1999; Lathem et al., 2005; Rogers et al., 2007; Russell et al., 2000; Saslaw & Carlisle, 1969; Sawyer et al., 1966; Welkos et al., 1995; Welkos et al., 1997). Based on the lowest log10 CFU detected for each of these organisms in the decontamination controls in this study, the concentration of viable bacteria recovered from each of the test materials falls within the respective estimated infectious dose range for each of these bacterial species. This suggests that, although the concentration of recovered viable bacteria for each species was observed

Table 2

Decontamination Efficacy of B. suis, B. pseudomallei, F. tularensis, Y. pestis and Biological Indicators Following Vaporous Hydrogen Peroxide Fumigation.

Organism/Test Materiala B. suis Glass Hypalon® Glove Stainless Steel B. pseudomallei Glass Hypalon® Glove Stainless Steel F. tularensis Glass Hypalon® Glove Stainless Steel Y. pestis Glass Hypalon® Glove Stainless Steel Biological indicators (decontaminated) Biological indicators (not decontaminated) aAll

No. Tested (No. Positive) Day 1 Day 7 3 (0) 3 (0) 3 (0)

3 (0) 3 (0) 3 (0)

3 (0) 3 (0) 3 (0)

3 (0) 3 (0) 3 (0)

3 (0) 3 (0) 3 (0)

3 (0) 3 (0) 3 (0)

3 (0) 3 (0) 3 (0) 16 (0) 1 (1)

3 (0) 3 (0) 3 (0) 16 (0) 1 (1)

blanks (not inoculated) for each material were negative for growth.

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to decrease significantly over a period of less than 24 hours, the number of viable microorganisms remaining on each of the test surfaces poses a potential infectious contact hazard requiring further decontamination. In fact, the results of this study show that glass, Hypalon® rubber glove, and stainless steel contaminated with B. suis, B. pseudomallei, F. tularensis, and Y. pestis and exposed to vaporous hydrogen peroxide were completely inactivated as demonstrated by the lack of observable growth after 7 days of incubation in liquid culture. In this study, the approach used to evaluate the effectiveness of vaporous hydrogen peroxide against B. suis, B. pseudomallei, F. tularensis, and Y. pestis was a combination of both quantitative and qualitative growth assessments. The quantitative approach enables the evaluation of decreases in the number of viable bacteria as a result of the initial drying of the inoculum as well as the length of time the non-decontaminated controls were maintained in parallel to the decontamination run. Although these quantitative values enable log-reduction determinations, the fact that coupon extraction procedures typically yield less than 100% recovery of microorganisms from test material coupons limits the quantitative data to providing a conservative estimate of the level of viable microorganisms remaining. Therefore, the decontamination efficacy observed in this study for vaporous hydrogen peroxide against B. suis, B. pseudomallei, F. tularensis, and Y. pestis should be based on the number of viable bacteria recovered from the decontamination controls, and not the inoculum or 1-hour drying controls. Such efficacy calculations would yield log reductions ranging from 3.2 to 3.4 for Y. pestis and 7.5 to 7.6 for B. pseudomallei. By combining the quantitative approach with the qualitative growth/no growth assessments, a better estimate of complete inactivation of microorganisms can be determined and potentially yield a more accurate representation of the viable bacterial load present as a function of persistence in the 1-hour drying controls and decontamination controls as well as decontamination efficacy. This approach has been used previously in evaluating the decontamination of Y. pestis in which a significant reduction in the log10 CFU of Y. pestis on 10 non-porous materials was observed between the 1-hour drying and decontamination controls (Rogers et al., 2008). This study demonstrates that vaporous hydrogen peroxide is an approach for inactivating B. suis, B. pseudomallei, F. tularensis, and Y. pestis dried on non-porous surfaces. It should be noted that this inactivation is based on the decontamination cycle parameters established for the ARCA chamber, and implementation of vaporous hydrogen peroxide fumigation for larger volumes such as rooms and buildings will require cycle parameter development specific for these areas. In fact, a recent study has demonstrated an approach in which cycle parameters can be developed and safely tested for inactivating biological agents with vaporous hydrogen peroxide (Richter et al., 2009). Such information would 30

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be applicable for conducting scheduled or unscheduled room decontamination with vaporous hydrogen peroxide. To date, no criterion is established for determining an appropriate level of decontamination of B. suis, B. pseudomallei, F. tularensis, and Y. pestis; however, complete inactivation is the current requirement for remediation of sites contaminated with Bacillus anthracis (Canter, 2005). The data presented in this study may help in addressing such a requirement; however, more data need to be collected in future studies that evaluate additional variables such as complex material types (e.g., porous surfaces), effects of temperature, and the presence of bioburden.

Acknowledgments This study was funded by Battelle’s Internal Research and Development Program. We thank Jennifer Price for her review of this manuscript.

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