Decontamination of materials contaminated with Bacillus anthracis and Bacillus thuringiensis Al Hakam spores using PES-Solid, a solid source of peracetic acid

Authors


Correspondence

Tony Buhr, Naval Surface Warfare Center – Dahlgren Division, CBR Concepts and Experimentation Branch, 4045 Higley Road Suite 345, Dahlgren, VA 22448-5162, USA. E-mail: DLGR_NSWC_Z20@navy.mil

Abstract

Aims

To develop test methods and evaluate survival of Bacillus anthracis Ames, B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores after exposure to PES-Solid (a solid source of peracetic acid), including PES-Solid formulations with bacteriostatic surfactants.

Methods and Results

Spores (≥7 logs) were dried on seven different test materials and treated with three different PES-Solid formulations (or preneutralized controls) at room temperature for 15 min. There was either no spore survival or less than 1 log (<10 spores) of spore survival in 56 of 63 test combinations (strain, formulation and substrate). Less than 2·7 logs (<180 spores) survived in the remaining seven test combinations. The highest spore survival rates were seen on water-dispersible chemical agent resistant coating (CARC-W) and Naval ship topcoat (NTC). Electron microscopy and Coulter analysis showed that all spore structures were intact after spore inactivation with PES-Solid.

Conclusions

Three PES-Solid formulations inactivated Bacillus spores that were dried on seven different materials.

Significance and Impact of the Study

A test method was developed to show that PES-Solid formulations effectively inactivate Bacillus spores on different materials.

Introduction

Chemical decontaminants can be effective sporicides. There is a need to develop new decontaminants with high efficacy against threat agents that is balanced with materials and equipment compatibility, minimal storage and transportation restrictions and ease-of-use for the consumer (Buhr et al. 2012a,b). Ideally, a decontaminant would also be low cost, readily available, safe for the environment and the user and manifest a small logistical footprint (Canter 2005).

Many current decontamination solutions are based on oxidative chemistry in aqueous systems. Elimination of water in premixed decontaminant formulations, resulting in reduced shipping weight and space requirements in the field, is a current goal of Department of Defense joint chemical and biological defense programs (Rowe and Murphy 2011). High test hypochlorite (HTH) and supertropical bleach (STB) are solid oxidizers currently listed in the field manual for decontamination (Anonymous 2006). Health risks associated with the use of chlorinated oxidizers include the release of toxic chlorine gas and the production of carcinogenic chlorinated organic compounds when mixed with ammonia or acids (Rutala and Weber 2008). HTH and STB are also hazardous chemicals with generally poor materials compatibility; they are known to corrode a number of metals (Craig and Anderson 1995; Garverick 1995). Annual corrosion costs in the United States were estimated to exceed $276 billion in 1998 (Koch et al. 2003). Corrosion-related costs and downtime for equipment maintenance, including ships, aircraft and vehicles (Herzberg 2012), justify the need to develop new decontaminants with reduced corrosivity.

As an alternative to hypochlorite salts, nonhalogenated peroxygen compounds such as peracetic acid are promising in decontamination and hazard mitigation applications (Bizzigiotti and Nickol 2012). Peracetic acid is of particular interest for biological decontamination as it is a well-known antimicrobial and sporicide (Baldry 1983; Block 2001; Rutala and Weber 2008). Peracetic acid can be generated in situ from a peroxygen source and an acyl donor, including the reaction of hydrogen peroxide and tetraacetylethylenediamine (TAED) (Block 2001; Coucharriere et al. 2002). PES-Solid (Solvay Chemicals, Inc., Brussels, Belgium) is a powder developed for the commercial detergent industry that releases peracetic acid directly and immediately upon dissolution in water (Raja et al. 2006; Rabe 2012). PES-Solid is a thermally stable, low-hazard solid oxidizer that is classified as peroxide Type G, approved for unregulated ground transport and not subject to organic peroxide restrictions during air or vessel transit in packages of up to 50 kg as indicated by the manufacturer and United States Department of Transportation case number CA2009090006 (Bundesanstalt für Materialforschung und –prüfung 2010; US Department of Transportation 2011; Rabe 2012).

The goals of this work were to develop test methods in order to characterize and quantify inactivation of Bacillus spores dried on porous and nonporous materials by solutions of PES-Solid with and without bacteriostatic surfactants. Test methods were developed to safely contain spores within single conical tube filter units throughout the entire test. The experimental design was driven by experimental controls, while method development was driven by practices intended to reduce human handling, reduce processing time, increase data output and increase both practical and statistical confidence of data output (Buhr et al. 2012b).

Materials and methods

Organisms and strains

Bacillus anthracis Ames, B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores were prepared in broth and characterized as previously described by Buhr et al. (2008, 2011, 2012a) using light microscopy, Coulter analysis and electron microscopy.

Decontaminant preparation

PES-Solid (Solvay Chemicals Inc., Brussels, Belgium) was added to surfactant formulations or sterilized water at 50 g l−1 and mixed for 15 min using a magnetic stirrer. Two surfactant systems were used: SSDX-12™ (TDA Research, Inc., Wheat Ridge, CO, USA) and the U.S. Navy's Dahlgren Surfactant System (DSS). SSDX-12™ was diluted 15 : 1 (water/concentrate) prior to use. DSS is a microemulsion that contains surfactants and was designed specifically for use with PES-Solid (US patent 7064241) and contained the following: 37·4 wt% dimethyldecylamine oxide surfactant (Lonza, Basel, Switzerland, Barlox 10S), 2·5 wt% ethoxylated tridecyl alcohol (Croda, East Yorkshire, England, Synperonic 13/6-LQ-AP), 26·3% glycerol (Fisher Scientific, Pittsburg, PA, USA #AC15892-0250) and 36·5 wt% water (Brown et al. 2006). Decontaminants were mixed immediately before use. For preneutralized controls, 25 ml of fresh decontaminant was mixed with 25 ml of 5% sodium thiosulfate (Sigma-Aldrich, St. Louis, MO, USA, S-7026) (STS) for 5 min on a magnetic stirrer immediately before covering spore-inoculated materials.

Coupon materials and sterilization

Coupons (2 cm2) of seven different test materials were inoculated with spores (7 logs was the target spore quantity) suspended in 0·1% Tween 80. Test materials included aluminium coupons painted with water-dispersible aircraft performance coating (APC), bare stainless steel 304, stainless steel 304 coated with Navy ship topcoat (NTC) and stainless steel 304 coated with CARC-W purchased from the coatings group at the University of Dayton Research Institute (UDRI), Dayton, OH. Magnesium fluoride-coated glass (MgF2 Glass) was purchased from Thermo Fisher Scientific, Advanced Glass Technology (Portsmouth, NH). Low-density polyethylene (LDPE) and GE Lexan® coupons were purchased from Laird Plastics (Baltimore, MD, USA).

Prior to testing, coupons were sterilized as previously described by Buhr et al. (2012a). However, due to its melting temperature, LDPE coupons could not be autoclaved. LDPE coupons were soaked in pH-adjusted bleach (0·6% hypochlorite, 0·2% acetic acid, pH = 7) for 10 min, followed by a 90% ethanol rinse. Sterilized coupons were stored in sterile containers until they were used.

Test method and design

A total of 245 replicates including all test samples and controls were tested for each Bacillus strain. A replicate was defined as an individual substrate (e.g. coupon or filter insert) inoculated with one of five independent spore preparations for each of the three Bacillus test strains. Three fresh decontaminant formulations (PES-Solid in water, SSDX-12™, or DSS) and three corresponding preneutralized decontaminant formulations were tested for each test run. Decontaminant-coupon contact time was 15 min. For each biosafety level 2 laboratory (BSL2) test run, five replicates of Banthracis ∆Sterne and five replicates of B. thuringiensis Al Hakam were tested for each substrate with both freshly prepared and preneutralized formulations. For each BSL3 test run, five replicates of Banthracis Ames were tested for each substrate with freshly prepared and preneutralized formulations. In addition, every test run included wet spore samples (spores suspended in 0·1% Tween 80 but never dried), which were considered the 100% spore recovery controls.

Figure 1 is a diagram of the decontamination test method. Spores were inoculated on test substrates as previously described (Buhr et al. 2012a). Additionally, wet spore controls consisted of Amicon® Ultra-15 Centrifugal Filter Units with Ultracel-100 membranes (Millipore, Billerica, MA, USA, UFC9010096) (filter units) containing 4·9 ml aqueous 0·1% Tween 80 and 0·1 ml spores (wet spores). The wet spore controls also contained 27 ml of aqueous 0·1% Tween 80 inside the conical tube below each filter unit to prevent the 0·1% Tween 80 in the filter inserts from dripping through the 100 000-nominal molecular-weight cut-off (MWCO) filter membrane. This prevented the spores from drying. Data from the wet spore controls represented the maximum possible number of recovered spores on a particular test day. Uninoculated, spore-free substrates were used as negative controls. In addition, 0·1 ml of the spore inoculum was serially diluted in 0·1% Tween 80 solution and immediately spread-plated on tryptic soy agar (TSA) to quantify inoculum titre on the day of substrate inoculation. TSA plates were incubated at 37 ± 2°C for 16 ± 2 h. Dried coupons were aseptically transferred to 50-ml conical tubes equipped with filter units, capped and stored at 22 ± 3°C.

Figure 1.

Step-by-step diagram of the PES-Solid decontamination method.

Exposure to fresh and preneutralized decontaminant

Test substrates were exposed to 2 ml of freshly prepared decontaminant added to spore-inoculated substrates at 15-s intervals. Two ml of 5% STS was added to each sample after 15 min of contact time to quench the reactivity of PES-Solid. Four ml of preneutralized decontaminant was added to the preneutralized control substrates. After all test substrates were neutralized and the preneutralized control substrates had 15 min of contact time, 10 ml of autoclave-sterilized water was added to all tubes to dilute the surfactant solutions prior to centrifugation.

Removal of neutralized decontaminant and quantification of spore survival

After the addition of water, all tubes were centrifuged at 3100 g for 10 min with maximum braking. The filter units retained all test substrates and spores while the neutralized decontaminant was collected in the conical tubes below the filter units. After centrifugation, filter units were removed using sterile forceps, the neutralized decontaminant filtrate was decanted, and the filter units were placed back in to the now-empty conical tube. Fifteen ml of autoclave-sterilized water was added to each filter unit and centrifuged again at 3100 g for 10 min with maximum braking. Water was used as a wash to solubilize and remove potentially bacteriostatic, residual surfactants and salt precipitates. After the second centrifugation, washed filter units containing test and preneutralized substrates were transferred into 50-ml conical tubes containing 27 ml autoclave-sterilized water. The original 50-ml conical tubes containing the filtrate were discarded. Ten ml of preheated (37°C) medium [1% glucose (G5767; Sigma, St Louis, MO, USA), 3% tryptic soy broth (TSB; T8907, Fluka, Buchs, Switzerland), 0·25% buffered peptone water (BPW;1·07228·0500, EMD, Darmstadt, Germany), 0·05% Tween 80 (BP338; Fisher, Pittsburgh, PA, USA), pH 7] was added to each filter unit and incubated at 37°C, 60 min for spore extraction. Five ml of preheated (37°C) medium [2% glucose, 6% TSB, 0·5% buffered peptone water] was added to the wet spore controls for a total of 10 ml and then incubated at 37°C for 60 min. After incubation, filter units were extracted by pipette mixing. The total time from the addition of extraction medium to plating was <90 min for each test run. Spore survival was quantified using dilution plating on TSA plates and scored for growth after incubation at 37 ± 2°C for 16 ± 2 h. The filter units with the remaining extraction medium, substrates and spores were also incubated at 37 ± 2°C for 16 ± 2 h to qualitatively assess the viability of all remaining spores, including those not removed from substrates.

Spore survival calculations

The wet spore controls served as the 100% recovery reference values for calculating spore survival after PES-Solid treatment and analysis. The average number of spores extracted from each preneutralized substrate control (i.e. dried spores on a substrate treated with preneutralized decontaminant) was divided by the average number from the wet spore controls in order to calculate spore recovery percentages for each spore–substrate pair.

The number of surviving spores (CFU ml−1) from each substrate was divided by the extraction percentage associated with that substrate to determine the number of surviving spores in CFU ml−1. Samples with positive growth in the filter unit, which contained the remaining extraction medium and the coupon with any remaining spores, but had no countable CFU on TSA were assigned 0·1 CFU ml−1 (1 CFU total in 8·8 ml). These samples were also corrected for extraction efficiency. The total number of surviving spores (CFU) for each test sample was then calculated by multiplying the surviving CFU ml-1 by 10 since there was 10 ml of extraction volume. A log10 transformation of the total surviving spores was performed (log10 (total CFU + 1)). Statistical means and standard deviations of the log-transformed spore survival data (CFU) were then calculated.

Chauvenet's criterion (Holman 2001) was applied to the raw data sets to identify outliers. For a data point to be identified as an outlier from a sample size of n = 5, the ratio of deviation from the mean to the sample standard deviation had to be greater than 1·65. The ratio was calculated using inline image where xi was the data point, xm was the arithmetic mean, and σ was the standard deviation (Holman 2001).

Transmission electron microscopy (TEM)

TEM analysis of untreated spores and PES-Solid-treated spores was conducted with greater than 109 spores as previously described (Buhr et al. 2008).

Results

Decontamination of substrates with PES-Solid® formulations

Five independent spore preparations were prepared for each Bacillus test strain. The mean titres just prior to spore harvest (preharvest titre), the mean titres after harvesting, centrifugation and resuspension in 10% of the sporulation medium volume (postharvest titres) and phase-bright percentages of heat-resistant spores are shown in Table 1. The average spore extraction percentages for each spore–substrate pair are shown in Table 2. The lowest extraction efficiency for all strains was from NTC. Bacillus thuringiensis Al Hakam spores were generally extracted with lower efficiency than B. anthracis spores.

Table 1. The mean spore titres (CFU ml−1) of heat-resistant (65°C, 30 min) spores and phase-bright percentage for five independent spore preparations of each strain
 Bacillus anthracis AmesBacillus anthracis ∆SterneBacillus thuringiensis Al Hakam
  1. a

    The titre was not directly quantified by plating and was calculated based on the titre after spore harvesting.

Titre before harvesting1·4 × 109a6·4 × 108 ± 4·3 × 1087·9 × 108 ± 1·7 × 108
Titre after harvesting1·4 × 1010 ± 3·3 × 1094·9 × 109 ± 1·0 × 1095·5 × 109 ± 1·2 × 109
Phase-bright spores (%)98·8 ± 0·497·0 ± 1·998·5 ± 1·2
Table 2. Spore extraction efficiency of Bacillus anthracis Ames, Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores
 B. anthracis Ames (%)B. anthracis ∆Sterne (%)B. thuringiensis Al Hakam (%)
  1. APC, aircraft performance coating; LDPE, Low-density polyethylene; NTC, Naval ship topcoat.

  2. a

    Wet spore controls were not centrifuged.

Water
CARC-W90·942·755·5
MgF2 glass70·236·022·6
Stainless steel 30458·741·620·0
APC76·038·716·7
NTC34·224·01·4
Lexan72·648·431·0
LDPE101·513·612·4
Weta100·0100·0100·0
SSDX-12
CARC-W91·066·015·4
MgF2 glass89·824·426·4
Stainless steel 30454·842·316·8
APC62·433·915·6
NTC29·019·81·4
Lexan59·449·022·5
LDPE 34·835·823·7
Weta100·0100·0100·0
Dahlgren surfactant system
CARC-W81·742·241·1
MgF2 glass84·227·09·2
Stainless steel 30456·429·96·1
APC91·041·713·3
NTC13·516·20·1
Lexan62·347·412·0
LDPE 55·635·712·8
Weta100·0100·0100·0

The log survival of spores following treatment with 50 g l−1 PES-Solid dissolved in three formulations (water only, SSDX-12™ diluted 1 part to 15 parts water and DSS) is shown in Table 3. Less than 1-log of viable spores was recovered for 56 of the 63 test combinations (strain/substrate/formulation) and less than 2-logs of viable spores for 61 of the 63 test combinations (Table 3). CARC-W and NTC were the only substrates with greater than 1-log of spore survival after PES-Solid treatment, but spore survival was limited to a maximum of 2·6 logs of survival.

Table 3. Log10 spore survival of Bacillus anthracis Ames, Bacillus anthracis ΔSterne and Bacillus thuringiensis Al Hakam spores on eight different substrates after a 15-min treatment with 50 g l−1 PES-Solid in three different solutions
 B. anthracis Ames (7·0 ± 0·3 logs coupon−1)B. anthracis ∆Sterne (7·2 ± 0·2 logs coupon−1)B. thuringiensis Al Hakam (7·2 ± 0·3 logs coupon−1)
  1. APC, aircraft performance coating; LDPE, Low-density polyethylene; NTC, Naval ship topcoat.

  2. a

    Four of five independent samples were 0·0 CFU, while 1 of 5 independent samples had some spore survival. Application of Chauvenet's principle indicates these samples are outliers, and the data sets could be treated as 0·0 ± 0.

50 g l−1 PES-Solid in water
CARC-W1·3 ± 0·81·6 ± 1·31·3 ± 1·6
MgF2 glass0·0 ± 00·4 ± 0·8a0·0 ± 0
Stainless steel 3040·3 ± 0·8a0·0 ± 00·0 ± 0
APC0·0 ± 00·0 ± 00·0 ± 0
NTC0·4 ± 0·9a0·1 ± 0·3a0·4 ± 0·8a
Lexan0·0 ± 00·0 ± 00·3 ± 0·3
LDPE0·0 ± 00·0 ± 00·0 ± 0
50 g l−1 PES-Solid in SSDX-12™ (Diluted 15 : 1 with water)
CARC-W1·9 ± 1·01·9 ± 1·22·2 ± 1·3
MgF2 glass0·0 ± 00·0 ± 00·0 ± 0
Stainless steel 3040·0 ± 00·0 ± 00·0 ± 0
APC0·1 ± 0·2a0·0 ± 00·0 ± 0
NTC0·5 ± 0·80·2 ± 0·3a0·7 ± 1·0
Lexan0·8 ± 1·10·1 ± 0·2a0·0 ± 0
LDPE0·0 ± 00·4 ± 0·8a0·1 ± 0·3a
50 g l−1 PES-Solid in the Dahlgren Surfactant System
CARC-W0·4 ± 0·60·0 ± 00·0 ± 0
MgF2 glass0·0 ± 00·0 ± 00·0 ± 0
Stainless steel 3040·0 ± 00·0 ± 00·0 ± 0
APC0·0 ± 00·0 ± 00·0 ± 0
NTC0·0 ± 00·7 ± 1·5a2·6 ± 1·5
Lexan0·0 ± 00·0 ± 00·0 ± 0
LDPE0·0 ± 00·0 ± 00·0 ± 0

The number of viable spores recovered from the PES-Solid-treated test substrates was statistically different (α = 0·05, P-value<0·05) from the corresponding preneutralized controls for 55 of the 63 test combinations (Table 4). In the 8 cases where the P-value was greater than 0·05, the preneutralized control had a standard deviation larger than the mean value (outliers were not removed for this t-test analysis). Importantly, the viable spores recovered from test substrates were significantly different for all B. anthracis-substrate pairings except the B. anthracis ∆Sterne-LDPE pairing, which was significantly different at α = 0·06. The remaining 7 cases where the P-value was greater than α = 0·05 were tests with B. thuringiensis Al Hakam spores. The lower statistical significance of B. thuringiensis Al Hakam results may be attributed to the extraordinary hydrophobicity/stickiness of this strain's exosporium (Buhr et al. 2012a and references therein).

Table 4. Calculated P-values resulting from independent samples t-tests (one-sided, left tail) at α = 0·05 between inactivated test replicates and the corresponding preneutralized controls
 Bacillus anthracis AmesBacillus anthracis ∆SterneBacillus thuringiensis Al Hakam
  1. APC, aircraft performance coating; LDPE, Low-density polyethylene; NTC, Naval ship topcoat.

  2. a

    P-values indicate differences were not statistically significant if P-value is >0·05.

  3. Test values were corrected for extraction efficiency.

50 g l−1 PES-Solid in water
CARC-W0·0017380·0000840·026508
MgF2 glass0·0074910·0036530·048527
Stainless steel 3040·0022410·0048310·116663a
APC0·0005400·0057670·029628
NTC0·0022500·0054920·047020
Lexan0·0009810·0036670·088854a
LDPE0·0025650·057618a0·019089
50 g l−1 PES-Solid in SSDX-12™ (Diluted 15 : 1 with water)
CARC-W0·0005620·0028020·049105
MgF2 glass0·0005500·0184390·116663a
Stainless steel 3040·0010450·0017360·071535a
APC0·0009990·0080460·043211
NTC0·0001550·0123080·133978a
Lexan0·0064390·0019440·023673
LDPE0·0137310·0081580·008205
50 g l−1 PES-Solid in the Dahlgren Surfactant System
CARC-W0·0014900·0071640·022875
MgF2 glass0·0022710·0125090·029628
Stainless steel 3040·0001380·0052170·047019
APC0·0005480·0216120·088854a
NTC0·0032160·0019300·149415a
Lexan0·0008100·0012600·029611
LDPE0·0027480·0052330·021394

Less than 1-log of spore survival was observed for 18 of 21 strain/substrate combinations treated with PES-Solid dissolved in water, 18 of 21 strain/substrate combinations treated with PES-Solid in SSDX-12™ and 20 of 21 strain/substrate combinations treated with PES-Solid in DSS (Table 3). Table 5 shows t-test comparisons at α = 0·05 for those substrates showing >1 log spore survival: CARC-W and NTC. Comparisons were made between formulations for each strain/substrate pair and between strains for each formulation/substrate pair. T-tests performed indicated that there were no statistically significant differences in spore inactivation between formulations or between strains (α = 0·05).

Table 5. Calculated P-values resulting from independent samples t-tests (two-sided) at α = 0·05 between inactivated test replicates corrected for extraction efficiency
 CARC-WNTC
  1. NTC, Naval ship topcoat.

  2. P-values >0·05 indicate differences that are not statistically significant.

  3. NA indicates no spore survival and thus no numerical differences in averages or standard deviations.

Bacillus anthracis Ames: formulation comparison
Water vs SSDX-120·222301NA
Water vs Dahlgren0·180597NA
Super soap vs Dahlgren0·180597NA
Bacillus anthracis ΔSterne: formulation comparison
Water vs SSDX-120·722494NA
Water vs Dahlgren0·525525NA
Super soap vs Dahlgren0·193205NA
Bacillus thuringiensis Al Hakam: formulation comparison
Water vs SSDX-120·5684580·177808
Water vs Dahlgren0·3910020·057669
Super soap vs Dahlgren0·3813310·064135
50 g l−1 PES-Solid in water: strain comparison
B. anthracis ΔSterne vs B. anthracis Ames0·219855NA
B. anthracis ΔSterne vs B. thuringiensis0·394300NA
B. anthracis Ames vs B. thuringiensis0·394782NA
50 g −1 PES-Solid in SSDX-12™ (15 : 1): strain comparison
B. anthracis ΔSterne vs B. anthracis Ames0·297311NA
B. anthracis ΔSterne vs B. thuringiensis0·686432NA
B. anthracis Ames vs B. thuringiensis0·560093NA
50 g l−1 PES-Solid in the Dahlgren Surfactant System: strain comparison
B. anthracis ΔSterne vs B. anthracis AmesNANA
B. anthracis ΔSterne vs B. thuringiensisNA0·057669
B. anthracis Ames vs B. thuringiensisNA0·057669

Electron microscopy and spore sizes

TEM images of B. anthracis ∆Sterne or B. thuringiensis Al Hakam spores that were treated with 50 g l−1 PES-Solid for 15 min are shown in Fig. 2. All spore structures including the exosporium appeared intact after PES-Solid treatment. There were no differences in spores exposed to PES-Solid for 1, 5 or 60 min (images not shown) compared with 15 min.

Figure 2.

Transmission electron micrographs of Bacillus anthracis ΔSterne (top) and Bacillus thuringiensis Al Hakam (bottom) spores treated, from left to right, with water, PES-Solid in water for 15 min, PES-Solid in SSDX-12™ for 15 min and PES-Solid in Dahlgren Surfactant System (DSS) for 15 min. Bar = 0·5 μmol l−1.

Spore sizes were measured with a Beckman Coulter Multisizer (Table 6). The control sample used for statistical mean size comparisons was viable spores suspended in water. There was a statistical difference in spore size between spores in water and spores treated with preneutralized formulations despite the fact that there was no practical difference in spore viability or spore structure. These statistical results were driven by the large population sizes. Logic dictated that practical similarities (viability and spore structure) are higher priority than statistical results when the population sizes were outside the limits of statistical methods (Buhr et al. 2011, 2012a). The calculated statistical values for samples treated with PES-Solid, with or without surfactants, were also greater than the critical value, indicating that every sample was statistically different from the control. Once again, the large sample sizes surpassed the limits of the statistical methods.

Table 6. Volume-equivalent spherical diameter of spores before and after treatments with PES-Solid and preneutralized (PN) PES-Solid
Bacillus anthracis ΔSterne
 Spores countedTest statisticaSpherical diameter (μm)
MeanMedianMode
Water60 8662·691·152 ± 0·1381·1451·151
PN PES-Solid54 44812·101·162 ± 0·1421·1541·151
PES-Solid 1 min51 1133·701·149 ± 0·1331·1441·151
PES-Solid 5 min56 01210·241·144 ± 0·1291·1381·138
PES-Solid 15 min52 49021·421·135 ± 0·1291·1301·126
PES-Solid 60 min51 07530·151·128 ± 0·1281·1221·103
PN SSDX-12™ + PES-Solid66 33322·811·171 ± 0·1591·1581·163
SSDX-12™ + PES-Solid 15 min66 38214·111·141 ± 0·1401·1321·126
PN DSS + PES-Solid7 36735·441·219 ± 0·1551·2061·163
DSS + PES-Solid 15 min68 4253·881·155 ± 0·1401·1471·151
Bacillus thuringiensis Al Hakam
 Spores CountedTest StatisticaSpherical Diameter (μm)
MeanMedianMode
  1. DSS, Dahlgren Surfactant System.

  2. a

    Dunnett's test was performed with spherical diameter data to determine critical statistical values for 2-sided tests at α = 0·05 (Dunnett 1964).

Water48 0382·691·250 ± 0·1861·2191·200
PN PES-Solid48 4414·331·245 ± 0·1721·2171·188
PES-Solid 1 min46 0847·011·242 ± 0·1641·2171·188
PES-Solid 5 min42 91917·371·230 ± 0·1671·2041·213
PES-Solid 15 min44 10319·081·228 ± 0·1641·2041·175
PES-Solid 60 min43 98018·961·211 ± 0·1661·1851·175
PN SSDX-12™ + PES-Solid57 94919·261·238 ± 0·1841·2091·200
SSDX-12™ + PES-Solid 15 min62 32644·421·202 ± 0·1671·1781·163
PN DSS + PES-Solid12 06182·601·578 ± 0·4261·7081·873
DSS + PES-Solid 15 min60 79449·121·198 ± 0·1561·1751·163

Comparison of spore size modes showed that all samples (preneutralized or PES-Solid treated) were very similar in size. These data suggested that PES-Solid had minimal impact on spore size. The sole exception was B. thuringiensis Al Hakam spores treated with preneutralized PES-Solid in DSS, where spores were larger in size. This sample also had an extraordinarily large test statistic value (Table 6). There is no obvious explanation for this single, reproducible anomaly.

Discussion

Many decontaminant formulations manifest both bactericidal and bacteriostatic activities, particularly those containing surfactants (Birnie et al. 2000, Walsh et al. 2003; Fraud et al. 2005). Due to the need to develop and test new chemical decontaminants with both bacteriostatic and bactericidal activities, there is also a need to develop reproducible and efficient sporicidal test methods. Within this effort, a test protocol was developed to determine the efficacy of chemical decontaminants with known bacteriostatic (surfactants were present in many test formulations) and/or bactericidal activities (PES-Solid). In order to accurately quantify spore inactivation, it was necessary to neutralize and remove decontaminant test chemicals from treated spores. Method development focused on cost, efficiency, reliability and the suitability of the method for both the biosafety levels 2 and 3 (BSL2 and BSL3) laboratories. In this case, the BSL3 laboratory has four layers of regulations: the Centers for Disease Control, U.S. Department of Defense, U.S. Navy and Naval Surface Warfare Center, Dahlgren.

Conical tubes with 100 000 MWCO filter inserts were used to remove all test chemicals including bacteriostatic surfactants, while retaining each test coupon and all spores within the filter insert throughout the entire method. This procedure removed the bacteriostatic effects caused by the test chemicals in order to eliminate false-positive/false-negative data and increase data confidence. The maximum spore recovery from filter units was quantified from solution controls and was used to calculate the extraction efficiencies for each substrate/formulation pair. Post-treatment spore survival was then corrected for these extraction efficiencies to avoid reporting inflated decontamination efficacy results. These methods reduced test time and handling steps and increased daily data output in comparison with methods with multiple washes and spore extractions (Sagripanti et al. 2007; Tomasino et al. 2008).

Complete inactivation of at least 7 logs of Bacillus spores was achieved on all substrates by at least one formulation containing PES-Solid at 50 g l−1. Independent sample t-test comparisons showed no difference in performance between formulations, indicating that PES-Solid is active in the presence of numerous and various synergistic chemicals.

There was no statistical difference in spore inactivation when comparisons were made between strains, indicating that BSL2 tests with Bacillus anthracis ΔSterne and/or B. thuringiensis Al Hakam may be conducted in place of many BSL3 tests in order to reduce test time and costs. This also supports B. thuringiensis Al Hakam as a suitable simulant for field testing.

Collectively, the results indicated that the coatings CARC-W and NTC were the most difficult military surfaces to decontaminate. These substrates were also the most difficult to assess and had the most unreliable spore extractions. CARC-W has intentional surface roughness (Bizzigiotti 2012), and NTC has a pliable, sticky surface which makes them uniquely challenging substrates for decontamination. In fact, spore recovery percentages were lowest for NTC for all strains (Table 1), and manifested exceptionally low recoveries (<5%) for B. thuringiensis Al Hakam spores. It is speculated that B. thuringiensis hydrophobicity (Doyle et al. 1984; Koshikawa et al. 1989; Husmark and Ronner 1990; Ronner et al. 1990; Faille et al. 2002) contributed to strong interactions with NTC. It is further speculated that spores may settle on top of one another in low spots on CARC-W and NTC providing protection to spores that are buried.

Spore size was not affected, and there was no microscopic evidence of structural damage to any of the spore protective structures after exposure to the aqueous formulations of PES-Solid. These data suggest that PES-Solid-treated, structurally intact spores did not succumb to osmotic stress after spore inactivation. These results are similar to those obtained when these strains were treated with the mild decontaminant hot, humid air (Buhr et al. 2012a). In contrast, Coulter analysis and TEM images of macrobacillus spores showed significant damage to the outer structures of spores following treatment with a chlorinated oxidizer, pH-adjusted hypochlorite (Buhr et al. 2008, 2012a).

Interestingly, the mechanism of peracetic acid oxidation has specificity for organic sulfides in contrast to hypochlorite, which has general chemical reactivity (Karunakaran and Kamalam 2000). The test conditions here were also at near-neutral pH, while hypochlorite is stored at high pH. These data suggest that PES-Solid is a less damaging decontaminant than hypochlorite, which may also translate to improved materials compatibility. The mechanism of PES-Solid-spore inactivation was not the focus of this work but may be addressed in future experiments.

Many chemical formulations used as decontaminants damage the materials they intend to clean and/or present hazards to users (Bizzigiotti and Nickol 2012; Smart 2012). Many of these formulations also present significant logistical burden and needs for precise preparation techniques, expensive, specialized equipment and/or extensive user training. Common solid oxidizers that are currently available are corrosive chlorine-containing materials. In a separate effort, 250 g L−1 PES-Solid in water was evaluated for compatibility with each of the materials used in efficacy testing (data not shown). There were no negative effects seen in any materials compatibility test conducted per ASTM standards (ASTM G 31–72, ASTM D 1003-00, ASTM D 543-95, ASTM F 502-02, ASTM D3363-92a, ASTM E 376-03, ASTM D2240-05). PES-Solid was also shown to be a nonirritant for erythema (redness) and oedema (swelling) in rabbit dermal testing when dissolved in water at 50 g l−1 (Snider and Babin 2008), the same concentration used here to inactivate Bacillus spores. Thus, PES-Solid is an effective option with a low logistics burden, acceptable materials compatibility and a low hazard to the user and the environment.

The work here established baseline performance data for PES-Solid decontamination of clean substrates contaminated with relatively clean spores. A future aim is to test PES-Solid spore decontamination where additives (dirt and organic debris) can be treated as independent variables and combined with clean spores prior to decontamination testing (Buhr et al. 2012a,b).

Acknowledgements

We are grateful for technical expertise and PES-Solid samples from Jürgen Rabe and his colleagues at Solvay Chemicals, Inc. We would also like to recognize TDA Research, Inc. for providing samples quantities of SSDX-12™. This effort was supported by the Defense Threat Reduction Agency Joint Science and Technology Chemical & Biological Defense Program (JSTO-CBD). Special thanks go to Glenn Lawson, Charles Bass, Mark Morgan, Matthew Hornbaker, John Crigler, Ed Prokop, Kathy Crowley, Chris Hooban, Wynn Vo, Michael Simulcik and Meredith Bondurant. Jerry Brown and Richard Hodge were responsible for selection of PES-Solid as a solid oxidizer for decontamination. More than a decade's worth of requirement changes were endured to develop effective PES-Solid decontaminant formulations with improved materials compatibility.

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