EPA 600/R-14/405 | May 2015 | www.epa.gov/research

Comparative Efficacy of Sporicidal Technologies for the Decontamination of Bacillus anthracis, B. atrophaeus, and Clostridium difficile Spores on Building Materials

Office of Research and DevelopmentNational Homeland Security Research Center

EPA/600/R-14/405 May 2015

Comparative Efficacy of Sporicidal Technologies for the Decontamination of Bacillus anthracis, B. atrophaeus, and

Clostridium difficile Spores on Building Materials

U.S. Environmental Protection Agency Research Triangle Park, NC 27711

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Disclaimer

The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development’s (ORD’s) National Homeland Security Research Center (NHSRC), funded and directed this work through an Interagency Agreement DW-21-9234401-0/1 with Edgewood Chemical Biological Center (ECBC). This report has been peer and administratively reviewed and has been approved for publication as an EPA document. The views expressed in this report are those of the authors and do not necessarily reflect the views or policies of the Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use of a specific product.

Questions concerning this document or its application should be addressed to:

M. Worth Calfee, Ph.D. National Homeland Security Research Center Office of Research and Development U.S. Environmental Protection Agency Mail Code E343-06 Research Triangle Park, NC 27711 919-541-7600

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Acknowledgments

Contributions of the following individuals and organization to this report are gratefully acknowledged:

United States Army – Edgewood Chemical & Biological Center Dr. Vipin Rastogi Lisa S. Smith

United States Environmental Protection Agency (EPA) Dr. Worth Calfee, Office of Research & Development (PI)

Peer reviewers Dr. Doris Betancourt – US EPA, Office of Research and Development Dr. Frank Schaeffer – US EPA, Office of Research and Development John Archer - US EPA, Office of Research and Development

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Executive Summary

The U.S. Environmental Protection Agency (EPA) Office of Research and Development is striving to protect human health and the environment from adverse impacts resulting from acts of terror by investigating the effectiveness and applicability of technologies for homeland security (HS)-related applications. This report summarizes the data generated under an interagency program between Edgewood Chemical Biological Center (ECBC) and EPA, entitled “Comparing the Efficacy of Liquid and Fumigant Technologies for the Decontamination of Bacillus anthracis Spores and Bacillus atrophaeus Spores on Building Materials”.

Comparative sensitivity (or resistance) of the spores of Bacillus anthracis (Ames), Clostridium difficile (American Type Culture Collection (ATCC) 43498), and B. atrophaeus (Dugway Proving Ground-prepared ATCC 9372) to three commercial sporicidal technologies (vaporous hydrogen peroxide (VHP), chlorine dioxide gas (CD), and pH-amended liquid bleach) was evaluated. Comparative decontamination efficacy of these technologies has previously been evaluated for building interiors by the US EPA’s Office of Research Development. However, until now, no direct side-by-side laboratory efficacy studies had been conducted to compare the relative resistance of the Dugway B. atrophaeus spores (also known as Bacillus atrophaeus or B.g.) to the resistance of Bacillus anthracis Ames spores or C. difficile spores. The main objective of this study was to evaluate the validity of using Dugway-prepared B. atrophaeus spores as a surrogate for spores of the Ames strain of B. anthracis in decontamination testing. A surrogate is considered suitable if its resistance to the test chemical is equal to or slightly greater than the resistance of the organism being modeled. A secondary objective was to determine the relative resistances of B. anthracis, B. atrophaeus, and C. difficile. Understanding the relative chemical resistance of Bacillus spores and C. difficile spores will enable prediction of sporicide performance against Bacillus spores based upon the vast body of hospital disinfection/decontamination data generated for C. difficile.

Small-size coupons of glass and pinewood (2 x 5 cm) were inoculated with ~7 logs of spores contained in a 50-µL aliquot of a spore suspension. Spore recoveries from glass coupons ranged between 10 and 25 % for C. difficile and 40 and 70 % for the two Bacillus spore types. The spore recovery from pinewood was significantly lower. For the two Bacillus spore types, the recoveries ranged between 25 and 40 %, for C. difficile the recoveries were typically ~5 % of the inoculum. Overall, >6 logs of spores were recovered from glass and pinewood for all three spore types, thus meeting the criteria for a 6 log dynamic range and allowing demonstration of decontamination efficacies up to “6 log reduction”. Sporicidal efficacy results demonstrate that for all three technologies, B. atrophaeus spores showed a resistance to decontamination comparable to the B. anthracis Ames spores on both glass and pinewood surfaces. Interestingly, while the C. difficile spores kill profile by bleach and CD gas was comparable to the other spore types on both glass and pinewood, sensitivity of this spore type to VHP was different on glass vs. pinewood. Log reduction (LR) values against C. difficile spores on glass coupons were <5, compared to >6.5 or near-complete kill at high dosages (2-3 hour exposure with 150 parts per million (ppm)) for the two Bacillus spore types. The efficacy data for pinewood were more variable, and the kill was incomplete (only three-four LR even after three hours (h) of exposure). Taken together, the data strongly support the contention that B. atrophaeus spores serve as a suitable surrogate for the pathogenic B. anthracis Ames spores when using these three decontamination technologies. Finally, the data are also consistent with the conclusion that sporicidal technologies are quite effective against spores of C. difficile. Additional work needs to be done to confirm our observation that VHP (450 parts per million by volume (ppmv)-h) on glass is only partially effective against

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C. difficile spores, and if true, this partial effectiveness may have significant influence on infection control in medical treatment facilities and spread of this hospital-acquired infection.

Summary of Results

pH-Amended Bleach When used as a sporicidal decontaminant, the recommended concentration of pH-amended bleach is 6000 ppm (US EPA, 2011). In this study (due to biosafety concerns), instead of spraying the bleach solutions, the coupons were covered with a 1-mL aliquot for 2, 5, 10, and 20 minutes. The tests were performed in the summer of 2012 (Phase 1) and were limited to two spore types, B. anthracis Ames and B. atrophaeus, and in the summer/fall of 2013 (Phase 2). In addition to Bacillus spores, spores of C. difficile were also included in Phase 2. After the intended contact times, the excess liquid and the coupon were immediately pipetted/transferred into a 20-mL recovery medium containing 0.5 % sodium thiosulfate. Efficacy values were >6.5 LR even within a two-min exposure on glass coupons and barely 2 to 3 LR even after a 20-min exposure on pinewood.

Chlorine Dioxide CD gas was used at a target concentration of 3000 ppmv, and coupons were exposed for 0.5, 1, 2, and 2.5 h. On glass coupons, LR values were >6.5 for all three spore types even with a 0.5 h (1500-ppmv-h dosage) exposure. No significant difference in the kill profile of the three spore types was evident, although minor differences were noted on the glass coupons. However, the LR values of 5 to 6 were observed after only 2.5 h exposure (7500-ppmv-h) when inoculated pinewood coupons were evaluated. Similar sporicidal efficacy of CD gas on pinewood has been observed previously, and this surface was concluded to be hard-to-decontaminate with this fumigant (Rastogi et al., 2010).

Vapor Hydrogen peroxide VHP was used at a target concentration of 150 ppmv, and the coupons were exposed for 0.5, 1, 2, and 3 h. On glass coupons, high efficacy (7 LR for Ames and 4.5 LR for C. difficile) was observed at sub-lethal exposure times for Ames and C. difficile spores, but LR values of 2-5 were observed for B. atrophaeus spores at similar exposure times. At high dosage (450-ppmv-h), the LR values against C. difficile spores were significantly lower compared to the other two Bacillus spore types. The efficacy measured as LR was <5 and kill kinetics were less rapid than the other two sporicidal agents.

Comparative Sporicidal Efficacy against Spores of B. anthracis and B. atrophaeus For all three technologies tested at two different times (summer of 2012 and summer/fall of 2013), B. atrophaeus spores were as resistant to kill as spores of B. anthracis Ames. No significant difference was observed in relative sensitivities of the two spore types to the tested technologies. In some cases, B. atrophaeus spores were slightly more resistant than the spores of B. anthracis Ames. Consequently, the data suggest that B. atrophaeus spores are appropriate surrogates for B. anthracis Ames spores for the three tested technologies.

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Comparative Sporicidal Efficacy against Spores of C. difficile and B. atrophaeus/B. anthracis With the exception of pH-amended bleach on glass, spores of C. difficile demonstrated comparable resistance (or sensitivity) to all three technologies relative to Bacillus species spores on both surface types. On glass, at fractional kill levels, C. difficile spores exhibited greater resistance than the other two spore types, for all three technologies tested.

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Contents Disclaimer ................................................................................................................................... iii Acknowledgments.......................................................................................................................... iv Executive Summary ........................................................................................................................ v Abbreviations/Acronyms ................................................................................................................ x

1.0 Introduction ........................................................................................................................1 2.0 Technology Descriptions and Test Matrices ......................................................................2

2.1 Technology Descriptions ........................................................................................... 2 2.2 Test Matrices for Decontamination Technologies ..................................................... 2

3.0 Summary of Test Procedures .............................................................................................4 3.1 Biological Agent ........................................................................................................ 4 3.2 Test Material Surfaces ............................................................................................... 5 3.3 Spore Stock Preparation and Coupon Inoculation ..................................................... 6 3.4 Decontamination with pH-Adjusted Bleach .............................................................. 6 3.5 Decontamination with Fumigants, CD Gas and VHP ............................................... 7 3.6 Spore Extraction and Quantification .......................................................................... 7 3.7 Recovery Efficiency................................................................................................... 8 3.8 Decontamination Efficacy ......................................................................................... 8

4.0 Quality Assurance/Quality Control ....................................................................................9 4.1 Instrument/Equipment Testing, Inspection, and Maintenance .................................. 9 4.2 Equipment Calibration ............................................................................................. 13 4.3 QC Results ............................................................................................................... 13 4.4 Audits ...................................................................................................................... 13 4.4.1 Performance Evaluation Audit ............................................................................ 13 4.4.2 Technical Systems Audit ..................................................................................... 14 4.4.3 Data Quality Audit .............................................................................................. 14

5.0 Results and Performance of Sporicidal Fumigants and Disinfectants .............................15 5.1 Spore QA/QC ........................................................................................................... 15 5.2 Spore Recovery ........................................................................................................ 15 5.3 Sporicidal Efficacy................................................................................................... 17 5.3.1 pH-Amended Bleach ........................................................................................... 17 5.3.2 VHP ..................................................................................................................... 17 5.3.3 CD Gas ................................................................................................................ 18 5.3.4 Overall Results (Phase 1) .................................................................................... 19 5.4 Spore Recovery ........................................................................................................ 20 5.5 Sporicidal Efficacy................................................................................................... 20 5.5.1 pH-Amended Bleach ........................................................................................... 20 5.5.2 CD Gas ................................................................................................................ 23 5.5.3 VHP ..................................................................................................................... 24 5.6 Summary of Phase 1 and Phase 2 Results ............................................................... 24

6.0 Discussion and Conclusions .............................................................................................26 6.1 Comparing Efficacy against B. anthracis and B. atrophaeus .................................. 26 6.2 Comparing Efficacy against Spores of Bacillus species and C. difficile ................. 26

References ................................................................................................................................27

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Figures Figure 3.1. C. difficile colonies on a plate (A) and spores (B) ...................................................... 5 Figure 3.2. Anaerobic jar with GasPak ........................................................................................... 5 Figure 5.1. Spore recovery from glass and pinewood coupons during Phase 1. .......................... 16 Figure 5.2. Efficacy of pH-Amended Bleach (6000 ppm) ............................................................ 17 Figure 5.3. Efficacy of VHP (150 ppmv) ..................................................................................... 18 Figure 5.4. Efficacy of CD (3000 ppmv) ...................................................................................... 19 Figure 5.6. Efficacy of pH-Amended Bleach on Three Spore Types on Glass Coupons ............. 22 Figure 5.7. Efficacy of pH-Amended Bleach on Three Spore Types on Pinewood Coupons ...... 22 Figure 5.8. Efficacy of CD Gas on Three Spore Types on Glass Coupons .................................. 23 Figure 5.9. Efficacy of CD Gas on Three Spore Types on Pinewood Coupons ........................... 23 Figure 5.10. Efficacy of VHP on Three Spore Types on Glass Coupons ..................................... 24 Figure 5.11. Efficacy of VHP on Three Spore Types on Pinewood Coupons.............................. 25

Tables Table 2.1 Decontamination Technology Descriptions .................................................................... 2 Table 2.2 Test Matrix for Three Decontamination Technologies ................................................... 3 Table 3.1 Test Materials .................................................................................................................. 6 Table 4.1 Data Quality Objectives for Test Measurements .......................................................... 10 Table 4.2 Sample Performance Criteria ........................................................................................ 12 Table 4.3 Performance Evaluation Audits .................................................................................... 13 Table 5.1 QA/QC of Three Spore Types ...................................................................................... 15

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Abbreviations/Acronyms

ATCC American Type Culture Collection B. anthracis Bacillus anthracis (Ames strain) B. atrophaeus Bacillus atrophaeus BHI-HT rain heart infusion agar with horse blood and taurocholate BSC biological safety cabinet BSL bio-safety level °C degree(s) Celsius C. difficile Clostridium difficile CBR chemical, biological, and radiological cm centimeter CRP Critical Reagent Program CSI ClorDiSys, Inc. CD chlorine dioxide CFU colony forming unit(s) DNA deoxyribonucleic acid ft2 square foot/feet ft3 cubic foot/feet EPA U.S. Environmental Protection Agency ECBC Edgewood Chemical Biological Center FIFRA Federal Insecticide, Fungicide, Rodenticide Act g gram h hour(s) HS homeland security HSRP Homeland Security Research Program L liter(s) LR log reduction M molarity m3 cubic meter(s) mg milligram(s) min minute(s) mL milliliter(s) µL microliter(s) NaOCl sodium hypochlorite NaOH sodium hydroxide NHSRC National Homeland Security Research Center NIST National Institute of Standards and Technology ORD Office of Research and Development PCR polymerase chain reaction ppm part(s) per million ppmv part(s) per million by volume PO Project Officer PLC programmable logic controller psi pounds per square inch QA quality assurance QC quality control QMP quality management plan

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RH relative humidity SD standard deviation sec second(s) SOP Standardized Operating Procedure STS sodium thiosulfate TSA tryptic soy agar VHP vaporous hydrogen peroxide

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1.0 Introduction

The U.S. Environmental Protection Agency’s (EPA’s) Homeland Security Research Program (HSRP) is helping protect human health and the environment from adverse impacts resulting from the release of chemical, biological, or radiological (CBR) agents. With an emphasis on decontamination and consequence management, water infrastructure protection, and threat and consequence assessment, the HSRP is working to develop tools and information that will help detect and quantify the intentional release of chemical or biological contaminants in buildings, water systems, or the outdoor environment; contain these contaminants; decontaminate buildings, water systems or the outdoor environment; and facilitate the treatment and disposal of hazardous materials resulting from remediation/cleanup activities.

As part of the above program, EPA investigates the effectiveness and applicability of technologies for homeland security (HS)-related applications by developing test plans that are responsive to the needs of the HSRP’s EPA Program Office and Regional partners, conducting tests, collecting and analyzing data, and preparing peer-reviewed reports. All evaluations are conducted in accordance with rigorous quality assurance (QA) protocols to ensure that high quality data are generated and that the results are defensible. EPA provides high-quality information that is useful to decision makers in responding and implementing appropriate technologies to mitigate consequences to the public resulting from CBR incidents.

The purpose of this study was to evaluate the decontamination efficacy of three decontamination approaches for three spore types (B. anthracis Ames, B. atrophaeus, and Clostridium difficile). Comparative efficacy data for B. anthracis and B. atrophaeus were gathered to evaluate the suitability of B. atrophaeus as a surrogate for B. anthracis in decontamination studies. Such a comparative body of information is critical in evaluating candidate technologies in field- and building-scale studies, where no greater than a bio-safety level (BSL)-1 spore-forming organism can be used. The spores of B. anthracis and B. atrophaeus are quite different with respect to hydrophobicity and presence of an exosporium, which is present in B. anthracis Ames and not present in B. atrophaeus. It is therefore imperative that a comparative set of data be available for the test lead in selecting appropriate surrogates for future studies.

Comparative efficacy data for B. anthracis and C. difficile were gathered in attempts to bridge the hospital disinfection-related and homeland security-related bodies of literature. For example, when data from the current study are considered together with results from previous C. difficile studies, predictions of the effectiveness of technologies widely used in hospitals against B. anthracis may be possible. These predictions may prove to be valuable for future decontamination research in either area. C. difficile infection is the most common cause of nosocomial infectious diarrhea (Cloud and Kelly, 2007; McFarland et al., 2007).

During Phase 1 of this investigation, sporicidal efficacy was determined for bleach, chlorine dioxide (CD) gas, and vaporous hydrogen peroxide (VHP) against spores of B. atrophaeus and the pathogenic agent it is used to model, B. anthracis Ames. In Phase 2, C. difficile (spores) were added, as a third organism, to the test matrix. Additional decontamination trials were conducted with the three spore-forming organisms to evaluate the efficacy of the candidate technologies against an important hospital clinical pathogen (C. difficile) and the two Bacillus species.

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2.0 Technology Descriptions and Test Matrices

2.1 Technology Descriptions Table 2.1 summarizes the three decontamination technologies evaluated in this investigation. Information is provided on the manufacturer, product name, chemical components and active ingredients. Note that Ultra Clorox Germicidal Bleach is registered as a disinfectant, but the pH-amended solution is not. Further details on the chemical composition, preparation, and decontamination application procedures are provided in Section 3.

Table 2.1 Decontamination Technology Descriptions

Decontaminant Product Name,

Vendor, and Location

Active Ingredients and Sporicidal

Chemical Components EPA

Registration

Chlorine Dioxide CD-Gas ClorDisysTrenton, NJ Chlorine dioxide gas 3000 ppmv chlorine dioxide gas 80802-1

VHP

Vaporous hydrogen peroxide

STERIS Corp. Mentor, OH

Vaporized form of hydrogen peroxide

150 ppmv vaporous hydrogen peroxide 35 % VHP and balance water vapors

58779-4

pH-Amended Bleach

Ultra CloroxGermicidal

Bleach Clorox

Professional Products Co. Oakland, CA

Sodium hypochlorite, hypochlorous acid

Sodium hypochlorite 6.15 %, sodium hydroxide <1 %; diluted with sterile distilled water; with 5 % acetic acid added to adjust the pH to 6.8 - 7.2

67619-8 (Clorox

disinfectant)

Chlorine dioxide (CD) was selected for testing because CD has been demonstrated to be effective against B. anthracis on building materials (Canter, 2005, Canter et al., 2005; Rastogi et al., 2009, 2010). Furthermore, CD gas has been proven to be virucidal, bactericidal, and sporicidal (Beuchat et al., 2004; Fukuyama et al., 1986; Rastogi et al., 2009, 2010). VHP was selected for its proven sporicidal effect (Canter, 2005) and its recent evaluations in building-scale studies (U.S.-EPA BOTE project, 2013). pH-Amended bleach (with pH 7.0+0.1) was selected for testing because this decontaminant has been demonstrated to be effective against B. anthracis on many surface materials, is readily available, and easily prepared using off-the-shelf chemicals. It has also been evaluated in recent building-scale studies (U.S.-EPA BOTE project, 2013).

2.2 Test Matrices for Decontamination Technologies In general, the operating test conditions selected (e.g., contact time, concentration) were based on previous similar B. anthracis efficacy tests (as described above), as well as manufacturers’ recommended parameters. The test matrices for the three technologies (pH-amended bleach, VHP, and CD gas) are shown in Table 2.2. Working stocks of each spore type were diluted to ~2-5 x 108 spores/mL in 0.01 %

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Tween® 80. In general, glass and pinewood coupons were inoculated with a 50-µL aliquot as seven droplets across the surface. The spores were allowed to dry overnight in a Class II bio-safety cabinet (BSC) under ambient conditions. The inoculated coupons were used within 24 hours of drying. The coupons were covered with a 1-mL aliquot of pH-amended bleach and after the desired contact times (2, 5, 10, and 20 minutes (min) ), the excess liquid and the coupon were both transferred to a tube containing 20 mL of 0.01% Tween® 80 solution containing 0.5 % sodium thiosulfate (STS) for quenching the active moiety. For evaluating the gases, the inoculated coupons were placed in Petri plates and exposed in a 0.2265- cubic meter (m3) fumigation chamber to CD gas (3000 ppmv) or VHP (150 ppmv) as previously described in detail (Rastogi et al., 2009, 2010). A set of four coupons in a Petri plate was withdrawn after each exposure time (0.5, 1, 2, and 2.5 h) for CD gas and (0.5, 1, 2, and 3 h) for VHP. The coupons were aerated for 5 min in the Class IIBSC before being transferred into 20 mL of 0.01 % Tween® 80 solution.

Table 2.2 Test Matrix for Three Decontamination Technologies

Phase #

Biological Agent Coupon types Concentration of fumigant or

volume applied Contact Time (h)

1 B. anthracis

Ames B. atrophaeus

Glass and pinewood Four each

150 ppmv VHP 3000 ppmv CD

1 mL of 6000 ppm pH-amended bleach

0.5, 1, 2, and 3 h 0.5, 1, 2, and 2.5 h

2, 5, 10, and 20 min

2

B. anthracis Ames

B. atrophaeus C. difficile

Glass and pinewood Four each

150 ppmv VHP 3000 ppmv CD

1 mL of 6000 ppm pH-amended bleach

0.5, 1, 2, and 3 h 0.5, 1, 2, and 2.5 h

2, 5, 10, and 20 min

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3.0 Summary of Test Procedures

Test procedures were performed in accordance with an approved Quality Assurance Project Plan (QAPP) for “Comparing the Efficacy of Liquid and Fumigant Technologies for the Decontamination of Bacillus anthracis Spores and Bacillus atrophaeus Spores on Building Materials”.

3.1 Biological Agent The B. anthracis spores used for this testing were prepared from a documented stock of the Ames strain in a BSL-3 facility located in building E-3150 of the U.S. Army’s Edgewood Chemical Biological Center (ECBC, Aberdeen Proving Ground, Edgewood, MD). The Ames strain was procured from Critical Reagent Program (CRP) Unified Culture Collection at Fort Detrick, MD) and is maintained and characterized by stringent procedures internal to ECBC. The spores were prepared on large sporulation plates as per the procedure outlined in the QAPP, and spore stocks were maintained in 0.01 % Tween® 80. Specifically, the spore lots were characterized prior to use by colony morphology, direct microscopic observation and determination of percent refractivity and percent vegetative bacterial cells. In addition, the number of viable spores was determined by colony count and expressed as colony forming units per milliliter (CFU/mL). Genotypic identity of the frozen stock was confirmed by genomic deoxyribonucleic acid (DNA) extraction and amplification of chromosomal and plasmid-borne markers by polymerase chain reaction (PCR). Prior to each testing, spore quality was determined by three approaches: >95 % spores, heat resistance to 65 degrees Celsius (ºC) (<0.2-log difference after 30 min) and HCl resistance (<2-log reduction following 2 min exposure and 2-6 log reduction after 5 min exposure to 2.5-N HCl; U.S. EPA Protocols, 2014).

The B. atrophaeus spores (American Type Culture Collection [ATCC] 9372) were procured from Dugway Proving Ground, UT, as dry powder. One gram of spores was suspended in 40 mL of 0.01 % Tween® 80 and enumerated (by dilution plating) to determine the titer. Working stocks were prepared by appropriate dilution using 0.01 % Tween® 80 and enumerated. The spore stock was characterized by approaches similar to those used and described above for the B. anthracis Ames spore stock. This stock was also found to be resistant to HCl and heat.

The C. difficile (ATCC 43498) spores were initially procured from a commercial source (ATS Lab, Eagan, MN). However, the titer of the spores procured from the ATS laboratory was inadequate, so the spores were therefore prepared in-house according to the ASTM E2839 protocol (ASTM 2011), with one exception. All protocol steps were followed with the exception of the final step of spore purification using HistoDenz, which was omitted. The rationale for omission of the last step was twofold: a) the crop harvested from the plate was 90-95 % spores; and b) no similar purification was performed for the other two Bacillus spore species, and a comparable spore quality of C. difficile was thought to be the most appropriate to include C. difficile in this study. C. difficile is a BSL-2 bacterial strain (Figure 3.1 A and B). C. difficile cells were grown in reinforced clostridial broth (ATCC medium 2107) at 37 ⁰C in an anaerobic environment (80 % nitrogen, 10 % carbon dioxide, and 10 % hydrogen). Anaerobic jars containing GasPak (BD, Franklin Lakes, NJ; GasPak 150 system, catalog number 260628) were used to culture the viable spores (Figure 3.2). Colonies were grown on BHI-HT agar plates (brain heart infusion agar with

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horse blood and taurocholate, AS-6463; Anaerobe Systems, Morgan Hill, CA). Single colonies were visible after 48-72 h of incubation. The spore quality was determined using the same approaches used for the other two spore types, heat resistance and HCl resistance (<2 log reduction after 10-min exposure to 2.5N HCl).

The working stocks of all three spore types were prepared in 0.01 % Tween® 80 solution stored at 2 to 8 °C, and used within four weeks after preparation.

Figure 3.1. C. difficile colonies on a plate (A) and spores (B)

Figure 3.2. Anaerobic jar with GasPak

3.2 Test Material Surfaces

Information on the coupons and sterilization approaches used for testing is summarized in Table 3.1. Coupons were cut to uniform length and width from a large piece of stock material. Coupons were prepared for testing by sterilization via autoclaving using a Getinge Vacuum Steam Sterilizer (Model # 533LS, Goteborg, Sweden). The selected materials, shown in Table 3.1, were based on both cost-effectiveness and minimization of physical alterations of the material. Autoclaving was

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done at a minimum of 15 pounds per square inch (psi) and 121 °C for 30 min. Sterilization is intended to minimize contamination by microorganisms other than those being evaluated.

Table 3.1 Test Materials

Material Lot, Batch, or ASTM No., or Observation

Manufacturer Supplier Name,

Location

Approximate Coupon Size,

width x length

Material Preparation

Glass Wire-reinforced

glass McMaster Carr,

Elmhurst, IL 2 x 5 cm Autoclave

Pinewood Structural Wood, Hem-fir, type II

Bowater, Greenville, SC 1.5 x 1.5 cm Autoclave

3.3 Spore Stock Preparation and Coupon Inoculation

Working spore stocks with a target titer of 2-5 x 108 spores/mL were prepared in 0.01 % Tween® 80. The stocks were used within four weeks of preparation, and new working stocks were preparedeach month. The spore stocks were stored in a refrigerator at 2-8 ºC and thoroughly mixed before use. An aliquot of 50 µL was inoculated across the surface by transferring seven droplets. The inoculated coupons were dried in a Class II BSC overnight under ambient conditions (a range of 18-25 ºC and 20-45 % relative humidity [RH]). The dried coupons were used within 24 h. For each data point, a set of four test replicates was included. Controls included a negative coupon with no inoculation and a positive control that was not exposed to sporicidal agent.

3.4 Decontamination with pH-Adjusted Bleach Bleach was prepared and pH-adjusted (as per the internal protocol) fresh on the day of testing. The pH-amended bleach consisted of bleach diluted in water with its pH adjusted by addition of acetic acid. Specifically, Ultra Clorox® Germicidal Bleach was used, which contains 6.15 % by weight sodium hypochlorite (NaOCl) and <1.0 % sodium hydroxide (NaOH) in aqueous solution. This product has a pH between 11 and 12 and a density of 1.08 to 1.11 grams (g)/mL. The pH was adjusted to 6.5 – 7.0 by the addition of 5 % acetic acid. The primary active decontaminating agent in this final solution is hypochlorous acid. The recipe for preparation of pH-amended bleach for use as a decontaminant was as follows:

• Prepare 5 % acetic acid solution by diluting 50 mL of glacial acetic acid to 1 L with steriledistilled water in a volumetric flask.

• Mix eight parts sterile distilled water, one part Ultra Clorox® Germicidal Bleach, and addan adequate volume of 5 % acetic acid to adjust the pH to 6.8 – 7.2.

The pH-amended bleach decontamination procedure was conducted as follows: a pipette was used to dispense a 1-mL aliquot of pH-amended bleach to the surface of four replicate coupons. No reapplication of bleach was administered. Following the desired contact times (2, 5, 10, and 20 min), the excess liquid and the coupon were both transferred to a tube containing 20 mL of 0.01 % Tween® 80 solution containing 0.5 % sodium thiosulfate for quenching the active moiety and

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subsequent spore recovery procedures. The spores were recovered by ten-min sonication and two-min vortexing. Recoveries from test and control coupons were then determined by dilution-plating onto tryptic soy agar (TSA) plates.

3.5 Decontamination with Fumigants, CD Gas and VHP The test chamber used in these fumigation studies was 0.2265 m3 (8 cubic feet [ft3]) in dimension (2 ft x 2 ft x 2ft). The test chamber was constructed by ClorDiSys, Inc. (CSI) from 316-grade stainless steel. The vendor supplied the qualification documentation for performance of the test chamber. The chamber had one window for observation, which was completely covered when used with CD gas, since CD is light sensitive. This system has been safely utilized to test a wide variety of decontamination gases and vapors. The chamber was equipped with temperature, RH, pressure, and CD sensors. Additionally, the test chamber was equipped with five antechambers for easy access and removal of Petri plates containing inoculated coupons. Each antechamber had inner and outer airlock doors.

After fumigation with 3000-ppmv CD gas or 150-ppmv VHP for a pre-specified time, one Petri plate was placed in one of the antechambers by opening and closing of the inner door. The Petri plate was removed by opening the outer door without affecting the exposure of other coupons. The chamber was filled with CD at a flow rate of 20 L/min, and the concentration of CD was maintained near constant by a programmable logic controller (PLC) sensor regulator installed in the generator. A circulation fan installed in the chamber mimicked the air circulation produced by fans in a commercial large-room decontamination process. Air circulation in the chamber ensured that the gas/vapor was evenly distributed over the exposed surfaces. The chamber temperature and RH were programmed, controlled, and monitored. For CD testing, exposure was performed at target settings of 75 % RH and 24 ºC (75 ºF). For VHP testing, RH was reduced to <35 %, and the vapor concentration was set at 150 ppmv. The chamber was operated in ‘exhaust on’ mode when using VHP vapors. After the specified exposure time, each Petri plate was withdrawn from the chamber through the antechamber. The coupons were aerated in a Class II BSC for one-two min, following which each coupon was transferred into a tube containing 20 mL of 0.01 % Tween® 80 solution.

3.6 Spore Extraction and Quantification For coupons inoculated with B. anthracis Ames and B. atrophaeus, the tubes containing control or test coupons were vortexed for two min. However, in the case of C. difficile, the tubes containing control and test coupons were sonicated for 10 ten min (Bransonic Sonicator, procedures described in Rastogi et al., 2009, 2010) and vortexed for two min. For all three spore types, the spores were diluted tenfold in 0.01 % Tween® 80 to 10-4 for positive controls and to 10-1 for the test samples prior to plating. A 100-µL aliquot was plated on two TSA Petri plates at 10-3 and 10-4 dilutions (control sets) and from 100 and 10-1 dilutions (test samples). Plates were incubated at 37 ºC for 18-24 h to allow colonies to grow. Plates from test samples were left for another day to allow for growth of slow growing injured but viable spores. Colony forming units (CFU) were counted, averaged from two replicate plates, and transformed into log (CFU) for each replicate coupon. Averages were computed for each set of four replicate coupons.

7

3.7 Recovery Efficiency The mean percent spore recovery from positive control samples was calculated using results from positive control samples (inoculated, but not decontaminated), by means of the following equation:

Mean % Recovery = [Mean CFUpc/CFUtiter] × 100

where Mean CFUpc is the mean number of CFU recovered from four replicate positive control samples of a single material, and CFUtiter is the number of CFU inoculated onto each of those samples. The value of CFUtiter is determined from the working stock on the day of testing. Spore recovery was calculated for each of the two coupon types with each of the three spore types.

3.8 Decontamination Efficacy Sporicidal efficacy was computed by subtracting the mean log (CFU) of the test from the log (CFU) of the control values, according to the equation below. Standard deviations were also computed to assess the variability.

Efficacy = Log10CFUc – Log10CFUt, CFUc = total colony forming units from control coupons, and

CFUt = total colony forming units recovered from test coupons.

Since the amount of spore contamination on surfaces and the types of surfaces needing treatment following an actual contamination incident are expected to vary widely, it is impossible to evaluate all conditions (spore load, waste acceptance criteria, etc.) likely to be encountered during decontamination activities. To address this challenge and allow comparison across sporicidal products or decontamination methods, a consistent challenge is posed to evaluate effectiveness. For example, a 7- log spore challenge (inoculation of material coupon surfaces with ~ 5 x 107 spores) was used across all tests and materials. Consistent with sporicidal efficacy tests used to register sporicides under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), the current study utilized the generally accepted criterion of 6- LR to consider an approach effective. Recovery of no viable spores following treatment was considered highly effective.

8

4.0 Quality Assurance/Quality Control

QA/QC procedures were performed in accordance with the Quality Management Plan (QMP) and the test/QA Plan. The QA/QC procedures and results are summarized below.

In the measurement of decontamination efficacy, experimental error or variability could be introduced by inaccurate measurement of volumes of suspensions of bacteria being applied. Also inaccurate measurement of test parameters (e.g., disinfection solution concentration) being manipulated in the testing can contribute to error in decontamination efficacy. The data quality objectives for test measurements provided in Table 4.1 limit the error introduced into the evaluation. Quantification of B. anthracis, B. atrophaeus or C. difficile in this evaluation does not involve the use of analytical measurement devices. Rather, the CFU were determined by dilution plating and recorded. Sample performance criteria are shown in Table 4.2. Standard operating procedures (SOPs) implemented by qualified, trained, and experienced personnel were used to ensure data collection consistency.

4.1 Instrument/Equipment Testing, Inspection, and Maintenance A maintenance schedule for laboratory equipment was required. The equipment needed for the evaluation was maintained and operated according to the quality requirements and documentation of the evaluation facility. Equipment includes BSCs, pipettes, incubators, and orbital shakers. However, there were no critical experimental parameters that must be calibrated for the BSCs and orbital shaker equipment. Pipettes were calibrated every six months. Only properly functioning equipment was used; any observed malfunction was documented and appropriate maintenance or replacement of malfunctioning equipment was performed. Daily, the laboratory staff checked the temperature of the incubator and the results of the daily check of the incubator were entered into a facilities data collection form. The incubators were calibrated semi-annually on a schedule maintained by the ECBC.

Prior to use and following the frequency specified in Table 4.1, all calibrated equipment was checked by the user to verify that the equipment was within calibration. This information was also documented on data sheets that included equipment name, serial number, model number, date calibration was performed, and date calibration was due. The test personnel manually recorded this information by initialing and dating at time of verification.

The facility has SOPs for the calibration of all instruments. A list of all instruments requiring calibration is maintained in a database and calibrations are scheduled by designated staff. All instruments used at the time of experimentation were verified as being certified, calibrated, or validated. Calibration of instruments was done at the frequency shown in Table 4.1. Any deficiencies were noted, and the instrument was adjusted to meet calibration tolerances and recalibrated within 24 h or replaced. If allowable tolerances were not being met after recalibration, additional corrective action was taken, including the replacement of the instrument.

9

Table 4.1 Data Quality Objectives for Test Measurements

Test Measurement Specifications Instrument Specification

Parameter to be

Measured Unit

Allowable Test

Measurement Tolerance

Instrument Instrument Calibration/ Certification

Instrument Calibration Frequency

Expected Instrument Tolerance

Corrective Action if Expected

Instrument Tolerance

Unattained

Volume (i.e., spike or dilution volume)

µL ± 10 % Micro- pipette

Micropipettes were verified as

calibrated at time of use by

supplier- pipettes are recalibrated by gravimetric evaluation of

pipette performance by

supplier

Every six months ± 10 %

Replace with calibrated and

sufficiently accurate

micropipette

Weight g ± 0.1 Balance

Balances are calibrated

monthly and annually serviced

under a PMA

Every 12 months ± 0.1 g

Replace with calibrated and

sufficiently accurate balance

pH pH unit ± 0.1 pH unit pH meter

Perform two- point calibration

with standard buffers that

bracket targeted pH every time of

use. Percent slope value

generated by pH meter must be >

90 % to pass calibration

Prior to use ± 0.1 pH

unit Recalibrate instrument

10

Table 4-1 (Continued)

Test Measurement Specifications Instrument Specification

Parameter to be

Measured Unit

Allowable Test

Measurement Tolerance

Instrument Instrument Calibration/ Certification

Instrument Calibration Frequency

Expected Instrument Tolerance

Corrective Action if Expected

Instrument Tolerance

Unattained

RH in chamber %

± 20 % full scale Hygrometer

NIST*-traceable

certification and/or checked against NIST-

traceable hygrometer

Once per quarter

± 0.5 % from 25 %

to 95 % over the

range of 5 °C to 55 °C

Replace with calibrated

and sufficiently

accurate hygrometer

Temperature °C ± 2 °C Thermo-

meter

Checked against NIST-

traceable thermometer

Once prior to testing

± 0.5 °C at 25 °C

Replace with calibrated

and sufficiently

accurate thermometer

Time h two seconds

(sec)/h Timer Check against

NIST-traceable standard

Once per quarter two sec/h-

Replace with calibrated

and sufficiently

accurate timer

Colony

Colony Forming

Unit (CFU)

100 % of colonies must

be counted QCount™

Calibrated once a year

Once per month

one - two small

colonies per plate

Re-Calibrate Instrument

*NIST = National Institute for Standards and Technology

11

Table 4.2 Sample Performance Criteria

Sample Number of Samples

Information Provided

Performance Criteria

Corrective Action if Performance Criteria are not

Attained

Spike Control - direct analysis of

the B. anthracis, B. atrophaeus, and C.

difficile stock suspension

One sample each day of use at time

zero

Calculate value of CFU in stock suspensions

± 1 log (1 x 107 to 1 x 109 CFU/mL)

Discuss results with the PO; identify and correct the cause of incorrect bacterial levels in the stock

suspension(s)

Procedural Blank - not inoculated with either B. anthracis, B. atrophaeus, or C.

difficile a

One at time-zero (after overnight

drying)

Controls for sterility and cross

contamination No observed CFU

Discuss results with the PO; identify and

remove source of contamination

Positive Control - inoculated with

either B. anthracis, B. atrophaeus, or C.

difficile (not exposed to

disinfectant but extracted)

Three included per disinfectant

concentration and material

Controls for percent recovery

Mean CFU ≥ 5 % and ≤ 150 % of spike control

Discuss results with the PO

Test Coupon- inoculated with

either B. anthracis, B. atrophaeus, or C. difficile, exposed to the disinfectant and

extracted at non-zero time points

Five at each non-zero time point per

disinfectant concentration and

material

Replicate coupons that yield results impacted by test

conditions

CFU value outside three standard

deviations (SDs) of the mean will be evaluated as an

outlier

Discuss results with the PO

a Blank = Laboratory blanks were extracted at time zero and procedural blanks were placed in the test chamber and extracted at the same time as the samples exposed to disinfectant.

12

4.2 Equipment CalibrationAll equipment (e.g., pipettes, incubators, BSCs) and monitoring devices (e.g., thermometer, hygrometer) used at the time of evaluation were verified as being either certified, calibrated, or validated.

4.3 QC ResultsQC efforts conducted during decontaminant testing included positive control samples (inoculated, not decontaminated), procedural blanks (not inoculated, decontaminated), laboratory blank (not inoculated, not decontaminated), and inoculation control samples (analysis of the stock spore suspension). All positive control results were within the target recovery range of 1 to 150 % of the inoculated spores, and all procedural and laboratory blanks met the criterion of no observed CFU for all threeorganisms. Inoculation control samples were taken from the spore suspension on the day of testing and seriallydiluted, plated, and counted to establish the spore density used to inoculate the samples. The spore density levels met the QA target criterion of 2 x 108 CFU/mL (±1 log) for all tests.

4.4 AuditsPerformance Evaluation Audit

Performance evaluation audits were conducted to assess the quality of the results obtained during these experiments. Table 4.3 summarizes the performance evaluation audits that were performed.No performance evaluation audits were performed to confirm the concentration of B. anthracis, B. atrophaeus, or C. difficile spores. Unlike chemical analytes, commercially available quantitative standards do not exist for these organisms. The control samples and blanks support the spore measurements.

Table 4.3 Performance Evaluation AuditsMeasurement Audit

ProcedureAllowableTolerance

ActualTolerance

Volume of liquid from micropipettes Gravimetric evaluation ± 10 % ± 0.57 %

Time Compared to independent clock ± two sec/h 0 sec/h

Temperature Compared to independent calibratedthermometer ± 2 °C ± 0.36 °C

Relative Humidity Compare to independent calibrated hygrometer ± 10 % ± 2 %

Balance Compared to independent calibrated weight sets ± 0.5 g ± 0.03 g

13

Technical Systems Audit Observations and findings from the technical systems audit were documented and submitted to the laboratory staff lead. These audits were conducted to ensure that the tests were being conducted in accordance with the test/QA plan and QMP. As part of the audit, test procedures were compared to those specified in the test/QA plan and data acquisition and handling procedures were reviewed. None of the findings of the audits required corrective action.

Data Quality Audit All of the data acquired during the evaluation were audited. The data were traced from the initial acquisition, through reduction and statistical analysis, to final reporting, to ensure the integrity of the reported results. All calculations performed on the data undergoing the audit were checked.

14

5.0 Results and Performance of Sporicidal Fumigants and Disinfectants

5.1 Spore QA/QC To determine the quality of spore working stock preparations prior to testing, aliquots of spores were heat-treated at 65 ºC for 30 min and enumerated before and after treatment. The CFU/mL in control and treated samples were comparable (<20 % difference; <0.1 log SD); Table 5.1). The microscopic analysis of spores showed >95 % phase-bright spores and high titer (~109/mL). The working stocks of spores with an approximate titer of 2 x 108/mL were prepared by appropriate dilution with 0.01 % Tween® 80.

Table 5.1 QA/QC of Three Spore Types Sample ID Controla Heat-Shockb Difference (%)c

B. anthracis Ames 8.89 8.88 1 B. atrophaeus 8.93 8.75 15 C. difficile 9.63 9.52 20 a. Values represent log (CFU) averages based on two replicate measurements.b. Spore stocks were exposed to 65 ºC for 30 min and then were enumerated.c. These were computed from total CFU from control and heat-treated samples.

Phase 1 (B. anthracis Ames and B. atrophaeus)

5.2 Spore Recovery Prior to decontamination testing, spore recovery was determined for glass and pinewood coupons. The spores were inoculated as an aliquot of 50 µL deposited in droplets of seven µL spread over a 2 x 5-cm surface area. The spores were dried overnight at room temperature in a Class II biosafety cabinet. The dried inoculated coupons were transferred into tubes containing 20 mL of 0.01 % Tween® 80 and vortexed for two min. The spores were enumerated by dilution plating on TSA plates. Figure 5.1 summarizes the spore recovery results. While >7 log spores were recovered from glass coupons, the spore recovery from pinewood coupons was <7 logs. In general, the spore recovery of B. atrophaeus (60 % from glass and 10 % from pinewood) was less than the spore recovery of B. anthracis Ames (>90 % from glass and 50 % from pinewood). However, recoveries from all materials exceeded 6 logs, allowing demonstration of up to 6 LR in decontaminant efficacy.

15

A.

B.

Figure 5.1. Spore recovery from glass and pinewood coupons during Phase 1. A. Log spores; and B. Percent Recovery

0

1

2

3

4

5

6

7

8

Titer Glass Pinewood Titer Glass Pinewood

B. anthracis Ames B. atrophaeus

Log

(CFU

)

Spore Types and Positive Controls

Spores Inoculated vs Recovered from Glass and Pinewood (Phase 1)

0

10

20

30

40

50

60

70

80

90

100

Glass Pinewood

Perc

ent o

f Ino

cula

ted

Coupon Types

Spore Recovery from Glass and Pinewood

B. atrophaeusB. anthracis Ames

16

5.3 Sporicidal Efficacy

pH-Amended Bleach The bleach efficacy results are summarized in Figure 5.2. On glass coupons, with the exception of the survival of a small number of B. atrophaeus spores at early time points, both spore types werecompletely inactivated within 10-20 min of exposure to 6000 ppm of pH-amended bleach. In contrast, <1-2 log spore inactivation is evident on pinewood, even after 20 min of exposure. The low efficacy of bleach on pinewood was expected, based on previously reported data (Tomasino et al., 2010; Wood et al., 2011; Rastogi et al., 2012). In general, the two spore types demonstrated similar trends in their response to the pH-amended bleach challenge. For B. atrophaeus, recoveries of viable spores were typically higher (i.e., lower efficacy) than those for B. anthracis Ames at fractional kill levels.

Figure 5.2. Efficacy of pH-Amended Bleach (6000 ppm)CT = concentration

VHPTest coupons were exposed to 150 ppmv of VHP for 30, 60, 120, and 180 min. The efficacy results are summarized in Figure 5.3. With the exception of some variability over exposure times, complete spore kill was observed only for B. anthracis Ames spores on glass coupons. As expected, intermediate treatment (fractional kill) with shorter exposure times is expected to display high variability. Complete kill was not achieved on pinewood coupons, even with 3 h of exposure. Overall, the two strains demonstrated similar trends with regard to viability following VHP exposure. Additional experimental runs may allow more robust statistically-based comparisons of the data.

0

1

2

3

4

5

6

7

8

T0 = 0 T1 = 200 T2 = 500 T3 = 1000 T4 = 2000

Log

CFU

Rec

over

ed

CT Values (Derived with 2, 5, 10, and 20 min Exposure Times)

Sporicidal Efficacy of Sodium Hypochlorite @ 6000 ppm Concentration against B. atrophaeus and B. anthracis Ames Spores

Glass B. atrophaeusGlass B. anthracis AmesPinewood B. atrophaeusPinewood B. anthracis Ames

17

CD GasEfficacy results for the CD tests are summarized in Figure 5.4. On glass, complete kill was achieved for both spore types within 30 min of exposure to CD gas. In contrast, on pinewood ~2-logs of B. atrophaeus spores were recovered after two h of exposure to 3000-ppmv CD gas, while no viable spores of B. anthracis Ames were recovered. Higher variability under these moderately effective (fractional kill) conditions was evident. From these data, the two spore types demonstrate similar sensitivities (or resistance) to CD gas, with B. atrophaeus being slightly more resistant than B. anthracis.

Figure 5.3. Efficacy of VHP (150 ppmv)

0

1

2

3

4

5

6

7

8

T0 = 0 T1 = 75 T2 = 150 T3 = 300 T4 = 450

Log

CFU

Rec

over

ed

CT Values (Derived with 30, 60, 120, and 180 min Exposure Times)

Sporicidal Efficacy of VHP @ 150 ppm Concentration against Bacillus atrophaeus and B. anthracis Ames Spores

B. atrophaeus GlassB. atrophaeus PinewoodB. anthracis Ames GlassB. anthracis Ames Pinewood

18

Figure 5.4. Efficacy of CD (3000 ppmv)

Overall Results (Phase 1)Numerous conditions can affect decontamination efficacy. During the current tests, an inoculation density of 7 logs per 10 square centimeters (cm2) (8-9 logs/square foot [ft2]) is a significant challenge. At this inoculation density, spore layering is likely, resulting in challenges to sporicide penetration and limits to mass transfer rates. While prediction of the spore concentration on surfaces expected to be encountered following an actual release is impossible, laboratory tests utilizing a coupon inoculation procedure with a lower spore titer may have produced differingresults. Nonetheless, the chosen inoculum titer and coupon size were selected to maximize ease of testing, ability to replicate experimental runs, while maintaining the 6 log dynamic range of possible efficacy results. Further, slight differences in the preparatory conditions of the two strains may have introduced unintended variability or differences in chemical resistance. Spore preparation purity may impact spore resistance to chemicals, yet these effects remain largely unknown. Similar to results of previous studies (Rastogi et al., 2009, 1010; Tomasino et al., 2010), high variability in decontamination efficacy was observed at mid efficacy (fractional kill) levels. For Phase 1, the trends in efficacy data for B. atrophaeus spores and B. anthracis Ames spores are similar in their response to the three tested technologies. The data appear to support the conclusions that the two spore types are similar with respect to resistance to pH-amended bleach, VHP, and CD gas exposure. The data therefore support the use of B. atrophaeus as a surrogate for B. anthracis Ames in decontamination studies.Future tests may consider a broader set of surfaces with numerous inoculation densities (higher and/or lower). The tests conducted here were performed with suspension deposition; additional tests with dry spore deposition may also be of interest for future studies.

0

1

2

3

4

5

6

7

8

T0 = 0 T1 = 1500 T2 = 3000 T3 = 6000 T4 = 7500

Log

CFU

Rec

over

ed

CT Values (Derived with 30, 60, 120, and 150 min Exposure Times)

Sporicidal Efficacy of CD Gas @ 3000 ppm Concentration against Bacillus atrophaeus and B. anthracis (AMES) Spores

B. atrophaeus Glass B. atrophaeus Pinewood

19

Phase 2 (B. anthracis Ames, B. atrophaeus and C. difficile)

5.4 Spore RecoveryIn Phase 2, the work was extended to include spores of C. difficile, in addition to the two Bacillusspecies used in Phase 1. The results are summarized in Figure 5.5 for both coupon types and the three spore types. As seen in Figure 5.5, spore recovery values were > 6 logs, and the percent recoveries were 20-65 % from glass for all three spore types. Spore recoveries from pinewood were >30 % for Ames and <10 % for the other spore types. The overall trend appeared to support the conclusion that compared to the two Bacillus spore types, C. difficile spore recoveries were significantly lower (Figure 5.5-A). In the case of the C. difficile tests, ten min sonication, in addition to two min vortexing, was included to improve spore recoveries.

5.5 Sporicidal EfficacyThe sporicidal efficacy was determined for pH-amended bleach, CD gas, and VHP as in Phase 1. To avoid cross-contamination, C. difficile work was conducted in a separate dedicated Class II biosafety cabinet. The sporicidal efficacy results are summarized in the following sections.

pH-Amended BleachSpores were exposed for 2, 5, 10, and 20 min, followed by spore extraction from the control set and test set. Figures 5.6 and 5.7 summarize the efficacy results on glass and pinewood, respectively. Results were similar to the results observed during Phase 1, as all spore types demonstrated similar and rapid kill kinetics within two min of exposure, and near complete-kill by 5 through 20 min of exposure (Figure 5.6). Figure 5.7 summarizes the kinetics of spore kill on pinewood. As seen in Phase 1, once again the two Bacillus spore types and C. difficile spores were comparably resistant to bleach. Only 2 to 3 LR was observed for all three spore types even after 20 min exposure to bleach.

20

A.

B.

Figure 5.5. Spore Recovery from Glass and Pinewood A. Percent recoveries and B. Log (CFU)

0

10

20

30

40

50

60

70

80

90

100

B. anthracis Ames C. difficile B. atrophaeus

Perc

ent o

f Ino

cula

ted

Spore Types

Recovery of Spores from Glass and Pinewood

Glass Pinewood

0

1

2

3

4

5

6

7

8

9

TITER GLASS PINEWOOD

LOG

(CFU

)

COUPON TYPES

Spore Recovery of Three Spore Types from Glass and Pinewood

B. anthracis Ames C. difficile B. atrophaeus

21

Figure 5.6. Efficacy of pH-Amended Bleach on Three Spore Types on Glass Coupons

Figure 5.7. Efficacy of pH-Amended Bleach on Three Spore Types on Pinewood Coupons

0

1

2

3

4

5

6

7

8

9

T1 T2 T3 T4

LOG

RED

UC

TIO

N

TIME (2, 5, 10, 20 Minutes)

Average Log Reduction of Three Spore Types on Glass Effected by 6000-ppm Bleach

B. anthracis Ames C. difficile B. atrophaeus

0

1

2

3

4

5

6

7

T1 T2 T3 T4

LOG

RED

UC

TIO

N

TIME (2, 5, 10, 20 Minutes)

Average Log Reduction of Three Spore Types on Pinewood Effected by 6000-ppm Bleach

B. anthracis Ames C. difficile B. atrophaeus

22

CD GasOn glass coupons (Figure 5.8), the kill kinetics for all three spore types were comparable, and >6-LR was observed within 30 min of CD gas exposure. This result is similar to the results observed in Phase 1 for CD gas and the two Bacillus spore types. The spore kill kinetics on pinewood (Figure 5.9) were slower (3-4 LR up to two hours of exposure). On pinewood, even though the LR values were slightly lower (1.0) after 2 and 2.5 hours for Ames spores, overall the data support the contention that the three spore types are comparably resistant (or sensitive) to CD gas.

Figure 5.8. Efficacy of CD Gas on Three Spore Types on Glass Coupons

Figure 5.9. Efficacy of CD Gas on Three Spore Types on Pinewood Coupons

0

1

2

3

4

5

6

7

8

T1 T2 T3 T4

LOG

RED

UC

TIO

N

TIME (0.5, 1, 2, 2.5 Hours)

Average Log Reduction of Three Spore Types on Glass Effected by 3000-ppm CD

B. anthracis Ames C. difficile B. atrophaeus

0

1

2

3

4

5

6

7

T1 T2 T3 T4

LOG

RED

UC

TIO

N

TIME (0.5, 1, 2, 2.5 Hours)

Average Log Reduction of Three Spore Types on Pinewood Effected by 3000-ppm CD

B. anthracis Ames C. difficile B. atrophaeus

23

VHPThe sporicidal efficacy in terms of log reduction on glass coupons is summarized in Figure 5.10.As seen in the figure, >6 LR was observed for both Bacillus spore types after 2 h of exposure to VHP. At fractional kill levels, i.e., 30 min exposure to VHP, B. atrophaeus spores appear to be slightly more resistant to VHP than B. anthracis Ames spores. As for C. difficile spores, even though 4 LR was observed within 30 min of exposure, no further kill was observed on prolonged exposure up to a period of 3 h. Dosages required for complete kill of the two Bacillus spores appear to be inadequate to achieve the same level of kill for C. difficile spores. On pinewood coupons (Figure 5.11), only 3 to 4 LR was observed for the two Bacillus speciesspore types, even after 3 h of exposure to VHP. The B. atrophaeus spores appear to be more resistant than the Ames spores at fractional kill level, i.e., 30 min of exposure to VHP. VHP efficacy appears to be equally or slightly more effective against spores of C. difficile, i.e., 5 LR after 3 h of exposure.

Figure 5.10. Efficacy of VHP on Three Spore Types on Glass Coupons

5.6 Summary of Phase 1 and Phase 2 Results

The sporicidal efficacy testing for three technologies, pH-amended bleach, CD gas, and VHP, was performed in two phases. Phase 1 was conducted in the summer of 2012 with two Bacillus spore types, and Phase 2 was conducted in the summer/fall of 2013 with two Bacillus spore types plus spores of C. difficile. For each technology, four dosages derived from a fixed concentration with four exposure times were selected to observe fractional kill and high kill. High variability at fractional kill exposures resulting from high inoculation density, spore layering, and poor penetration of fumigant/disinfectant was observed, but these results are consistent with other studies.

0

1

2

3

4

5

6

7

8

T1 T2 T3 T4

LOG

RED

UC

TIO

N

TIME (0.5, 1, 2, 3 Hours)

Average Log Reduction of Three Spore Types on Glass Effected by 150-ppm VHP

B. anthracis Ames C. difficile B. atrophaeus

24

For pH-amended bleach, in both phases, the disinfectant was very effective on glass surfaces. In contrast, this technology (in both phases) was found to be less effective on pinewood. The efficacy data suggest the similarity in resistance of the two spore types, B. anthracis Ames and B. atrophaeus. Therefore, for bleach-based decontaminations, B. atrophaeus spores could serve as an appropriate surrogate for B. anthracis Ames. C. difficile demonstrated slightly higher resistance to pH-amended bleach on glass surfaces. These data suggest that hypochlorous acid-based technologies that have previously demonstrated effectiveness against C. difficile may have even greater efficacy against B. anthracis for hard, nonporous materials.

Figure 5.11. Efficacy of VHP on Three Spore Types on Pinewood Coupons

The efficacy of CD gas in both phases also demonstrates comparable effectiveness against spores of B. anthracis Ames and B. atrophaeus on glass coupons. Within 30 min of exposure, the LR values of >6 logs were observed. Overall, spore kill was more difficult on pinewood surfaces, but comparable sensitivity of the three spore types was evident in this study. Again, the data support the use of B. atrophaeus as a surrogate for B. anthracis in decontamination studies using CD, and previously demonstrated CD technologies for C. difficile may be promising for B. anthracis kill.

For VHP, variability in the efficacy at the fractional level was evident in both phases. On glass coupons, high dosages resulted in >6 LR for spores of both Bacillus species, but at fractional levels, B. atrophaeus appeared to be just as resistant (Phase 1) or slightly more resistant (Phase 2) than B. anthracis Ames. Interestingly, high dosages of VHP against C. difficile spores were only partially efficacious (LR ~5 for both materials). On pinewood coupons, high dosages in both phases appear to be partially efficacious, LR of only 3 to 4 for both Bacillus species. Overall, both Bacillus spore types demonstrated similar sensitivities to VHP, in both phases.

0

1

2

3

4

5

6

7

T1 T2 T3 T4

LOG

RED

UC

TIO

N

TIME (0.5, 1, 2, 3 Hours)

Average Log Reduction of Three Spore Types on Pinewood Effected by 150-ppm VHP

B. anthracis Ames C. difficile B. atrophaeus

25

6.0 Discussion and Conclusions

The main objective of the current study was to assess relative resistance (or sensitivity) of spores of B. atrophaeus (common surrogate for B. anthracis) and the spores of pathogenic B. anthracis Ames to three sporicidal technologies. Spores of the two Bacillus species were exposed to four dosages of pH-amended bleach, CD, and VHP. These technologies have commonly been employed in a number of previous cleanup efforts and have been evaluated in recent building cleanup demonstrations (US-EPA BOTE, 2013). The three technologies have been found to be very effective against both Bacillus species spore types on various surfaces, although numerous conditions can affect their efficacy. The choice of a given technology for decontamination must therefore consider the type of material surface being decontaminated.

6.1 Comparing Efficacy against B. anthracis and B. atrophaeus Are the two spore types significantly different in their sensitivity to decontamination technologies? Difference in sensitivity to decontamination technologies has been the premise for some critics to challenge studies using surrogate B. atrophaeus spores. Presence of an exosporium on Ames spores and their hydrophobicity could render such spores more resistant than the surrogate spores. If the surrogate spores were more sensitive than Ames spores, one would predict a faster kill rate and a significantly lower dosage required to achieve a complete kill of the surrogate organism, Bacillus atrophaeus. The present study was designed to address and investigate just these predictions. The results show a comparable kill rate and similar dosages required to achieve complete kill by all three decontamination technologies. The data therefore strongly favor the conclusion that the two spore types are comparable in their resistance to the three decontamination technologies evaluated. Taken together, the data support the use of B. atrophaeus spores as a surrogate for B. anthracis Ames in decontamination studies with these three technologies.

6.2 Comparing Efficacy against Spores of Bacillus species and C. difficile How different are C. difficile spores in their sensitivity to sporicidal chemicals relative to the other two Bacillus spore types? In general, the spores of C. difficile appear to adhere to the glass surface more tightly than the other two spore types. The spores of C. difficile also appear to be more resistant to pH-amended bleach at fractional kill levels. Complete kill even after a 20-min exposure time was not observed. These data suggest that hypochlorous acid-based technologies that have previously demonstrated effectiveness against C. difficile may have even greater efficacy against B. anthracis for hard, nonporous materials. Spores of C. difficile appear to demonstrate comparable resistance (or sensitivity) to the other two sporicidal technologies, i.e., CD gas and VHP. Considering the data collectively, technologies that have previously shown high efficacy against C. difficile spores may perform well against Bacillus spores. These data may allow targeted testing of other sporicidal decontaminants against Bacillus anthracis spores, based upon the results of C. difficile tests reported in hospital disinfection-oriented peer-reviewed journals.

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