Downloaded from on March 6, 2020 by guest€¦ · 1 1 A Standard Method to Inactivate Bacillus...
Transcript of Downloaded from on March 6, 2020 by guest€¦ · 1 1 A Standard Method to Inactivate Bacillus...
1
A Standard Method to Inactivate Bacillus anthracis Spores to Sterility Using γ-Irradiation 1
Christopher K. Cote1, Tony Buhr
2, Casey B. Bernhards
3,4, Matthew D. Bohmke
2, Alena M. 2
Calm3, Josephine S. Esteban-Trexler
1, Melissa C. Hunter
1, Sarah E. Katoski
3, Neil Kennihan
2, 3
Christopher P. Klimko1, Jeremy A. Miller
1, Zachary A. Minter
2, Jerry W. Pfarr
3, Amber M. 4
Prugh3, Avery V. Quirk
1, Bryan A. Rivers
4, April A. Shea
1, Jennifer L. Shoe
1, Todd M. Sickler
3, 5
Alice A. Young2 David P. Fetterer
1, Susan L. Welkos
1, Joel A. Bozue
1, Derrell McPherson
2, 6
Augustus W. Fountain III3, and Henry S. Gibbons
3* 7
1Bacteriology Division, United States Army Research Institute of Infectious Diseases, Fort 8
Detrick, MD 9
2Naval Surface Warfare Center, Dahlgren Division, Dahlgren, VA 10
3United States Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD 11
4Defense Threat Reduction Agency/National Research Council Research Associate Program, 12
Aberdeen Proving Ground, MD 13
Acknowledgements 14
15
We thank Angelo Madonna and the members of the Bacillus anthracis Irradiation and Viability 16
Technical Working Groups for valuable discussions during the design of the study. We thank 17
Mark Karavis and Alvin Liem (ECBC) for assistance with whole-genome sequencing and 18
informatics. Funding was provided by the Office of the Deputy Assistant Secretary of Defense 19
for Chemical and Biological Defense and by the Defense Threat Reduction Agency/National 20
Research Council Post-doctoral Fellowship Program (to C.B.B.) 21
AEM Accepted Manuscript Posted Online 13 April 2018Appl. Environ. Microbiol. doi:10.1128/AEM.00106-18This is a work of the U.S. Government and is not subject to copyright protection in the United States.Foreign copyrights may apply.
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
2
22
Abstract 23
In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped 24
preparations of γ-irradiated spores of Bacillus anthracis that contained live spores. In response, 25
a systematic, evidence-based method for preparing, concentrating, irradiating, and verifying 26
inactivation of spore materials was developed. We demonstrate consistency of spore 27
preparations across multiple biological replicates and show that two different DoD institutions 28
arrive independently at comparable dose-inactivation curves for a monodisperse suspension of B. 29
anthracis spores containing 3 × 1010
colony forming units (CFU). Spore preparations from three 30
different institutions in three strain backgrounds yielded similar decimal-reduction (D10) values 31
and irradiation doses required to assure sterility (DSAL) to the point at which the probability of 32
detecting a viable spore is 10-6
. Furthermore, spores of a genetically-tagged strain of B. 33
anthracis Sterne were used to show that high densities of dead spores suppress the recovery of 34
viable spores. Together, we present an integrated method for preparing, irradiating, and 35
verifying the inactivation of spores of B. anthracis for use as standard reagents for testing and 36
evaluating detection and diagnostic devices and techniques. 37
Importance: The inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of 38
live Bacillus anthracis (anthrax) spores to U.S. and international destinations revealed the need 39
to standardize inactivation methods for materials derived from Biological Select Agents and 40
Toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of 41
such an event. Following a retrospective analysis of the procedures previously employed to 42
generate inactivated B. anthracis spores, a study was commissioned by the DoD to provide the 43
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
3
data required to support the production of inactivated spores for the biodefense community. The 44
results of this work are presented in this publication, which details the method by which spores 45
can be prepared, irradiated and tested such that the chance of finding residual living spores in 46
any given preparation is 1/1,000,000. These irradiated spores are used to test equipment and 47
methods for detection of agents of biological warfare and bioterrorism. 48
Introduction 49
Like other species of the genus Bacillus, Bacillus anthracis, the causative agent of the disease 50
anthrax, forms hardy, metabolically dormant endospores upon exhaustion of nutrient supplies. 51
These spores are highly resistant to desiccation, temperature extremes, predation, exposure to 52
ultraviolet light, and ionizing radiation, and can survive for extended periods in conditions 53
unfavorable to the survival of vegetative bacteria (1-3). The environmental persistence, overall 54
hardiness, and the relative simplicity of production methods also have made B. anthracis a 55
favorite amongst nations that have historically pursued biological warfare including the United 56
States (pre-1972), the former Soviet Union, Iraq, and several other states that have been 57
suspected of harboring biological weapons programs (4, 5). 58
Because of its known history as a biological weapon, B. anthracis spores have also been utilized 59
as instruments of bioterrorism, notably by Aum Shinrikyo (6) and by the perpetrator of the 2001 60
anthrax attacks (7, 8) through the United States Postal Service. Therefore, development of 61
methods to detect and quantify B. anthracis spores in samples has been of great interest to the 62
public health and military sectors. However, the rigid, relatively impermeable spore structure (9-63
11) also renders the spores refractory to various molecular and biochemical detection methods 64
(12), as harsh mechanical disruption is required to extract nucleic acids in particular, 65
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
4
complicating the development of detection modalities. In addition to the intrinsic physical 66
properties of the spores, costly Biosafety Level-3 (BSL-3) procedures are generally required 67
during the development and testing of new detection and decontamination methods. Therefore, 68
methods of inactivating spores without destroying their physical and molecular characteristics 69
are highly desired and have been utilized over the past decades (13-16). 70
Methods for inactivating spores have historically included wet heat, chemical treatment methods, 71
and irradiation, many of which are reviewed elsewhere (13, 16). Wet heat forces steam under 72
high pressure through the otherwise relatively impermeable spore envelope, resulting in the 73
denaturation of protein components. As a result, wet heat methods compromise the integrity of 74
epitopes recognized by antibody-based detection methods (17, 18). Likewise, chemical methods 75
of decontamination (primarily using bleach, hydrogen peroxide, or ethylene oxide) result in 76
extensive free-radical damage that destroys important components of the spore, preventing 77
germination (19, 20). Because of its high penetrating power and more selective effects on large 78
macromolecular targets (e.g. DNA), γ-irradiation has become a favored method of inactivating 79
biological materials in implantable medical devices, food, and in the sterilization of laboratory 80
equipment (21-25), as irradiation can reduce the risk of contamination and provide assurance of 81
sterility with minimal remaining risks. 82
γ-irradiation was first reported as a suitable method for irradiating B. anthracis spores in 1959 83
(14). Subsequent work showed that spores of various Bacillus species typically exhibited 84
decimal reduction values (D10, the dose required to reduce the population by one log) of between 85
1.8 and 5.5 kGy, with marginal effects on spore ultrastructure, physical properties, and the 86
response to antibody- or PCR-based detection assays (13, 18, 26-29). Because of the perceived 87
efficacy of irradiation in yielding a safe yet responsive product that could mimic the essential 88
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
5
properties of live agent, irradiated spores of B. anthracis became a critical reagent for the 89
development of systems and devices to detect agents of biological warfare and bioterrorism. 90
Many of the experiments describing irradiation procedures, doses, and determination of D10 91
values for B. anthracis were summarized in the US DoD’s analysis of the inadvertent shipments 92
of live, virulent B. anthracis spores to unregistered laboratories in 2015 (30). However, as the 93
DoD report indicated, conditions under which the spores were grown, prepared, concentrated, 94
and handled were inconsistent from one experiment to another, preventing the direct comparison 95
of these data. Thus, the need for reproducible, standardized methods for producing such 96
materials was highlighted. 97
In many inactivation procedures, the risk that live organisms are present following sterilization is 98
approximated by the parameter known as the Sterility Assurance Level (SAL)(31). For medical 99
devices and pharmaceutical products sterilized with radiation, acceptable SALs range from 10-3
100
to 10-6
, depending on the sensitivity of the product itself to the effects of ionizing radiation (24). 101
The SAL represents the probability that a viable organism will be found in a sample subjected to 102
a given dose of radiation (e.g. an SAL of 10-6
indicates that one sample in 106 identically treated 103
samples will contain a viable organism). The SAL is typically extrapolated from the linear 104
portion of dose inactivation curves (“kill curves”) generated with a defined bioburden — usually 105
an appropriate, well-characterized, surrogate organism (25) — and depends on the linearity of 106
the inactivation curve up to the point of complete inactivation. To our knowledge, the concept of 107
the SAL has not been applied to inactivation of B. anthracis spore preparations or to the 108
preparation of biological materials per se (14, 15, 18, 19, 26, 32, 33), particularly for DoD 109
laboratories charged with preparing and distributing inactivated materials (30). 110
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
6
In this work, we present a method for preparing and inactivating B. anthracis spores to a SAL of 111
10-6
using γ-irradiation. In an inter-laboratory comparison performed in parallel at the United 112
States Army Edgewood Chemical Biological Center (ECBC) and the United States Army 113
Medical Research Institute of Infectious Disease (USAMRIID), we establish dose-inactivation 114
curves and decimal reduction values for multiple biological replicates using two different 115
irradiation platforms and use those data to extrapolate radiation doses sufficient to inactivate 116
spores of attenuated B. anthracis Sterne and fully virulent B. anthracis Ames to an SAL of 10-6
. 117
Finally, we characterize and define methods of detecting low levels of viable spores amidst a 118
background of inactivated spores. If followed stringently, the procedure for preparing and 119
irradiating spores presented here should prevent any further inadvertent distribution of live B. 120
anthracis spores and ensure that distributing laboratories remain in compliance with laws and 121
regulations governing biological select agents. 122
123
Results 124
Each independent spore preparation was completed on an independent date and surpassed the 125
quality/quantity requirements stipulated for preparation of B. anthracis spores as a standard 126
reagent for biodefense research. These requirements are delineated in Table 1. We reasoned that 127
each spore preparation should be derived from a well-sporulating parent strain with a known 128
passage history relative to the original isolate. Ideally, spore cultures should exhibit final spore 129
titers of at least 1 × 108 CFU/ml of sporulation medium (target is 1 × 10
9 CFU/ml) prior to spore 130
harvest and purification. The entire spore preparation was not heat shocked, but an aliquot was 131
removed for heat shocking. An aliquot of at least 1 × 107 spores from each spore preparation 132
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
7
exhibited heat resistance (65 °C, 30 min) using a standard quantitative plate assay. The spore 133
mode size should be consistently between 1.0-1.5 µm volume-equivalent spherical diameter 134
determined using a Beckman-Coulter Multisizer (BCM) (Figure 1). Spores were at least 95% 135
pure as judged by light microscopy counting of at least 100 particles per spore preparation 136
(Figure 1, inset). Spore dispersion was confirmed by examining at least 100 spores with light 137
microscopy (Figure 1, inset) and at least 500 spores with particle analysis via the BCM. The 138
BCM measurements of three independent preparations showed reproducible, single, uniformly 139
distributed particle size peaks demonstrating that there was no detectable spore clumping. 140
141
Determination of inactivation kinetics and sterilizing dose of B. anthracis - To determine the 142
batch-to-batch variability of radiation sensitivity and whether the irradiator configuration affects 143
inactivation, we conducted dose-inactivation studies on 3 ml aliquots of three independently 144
prepared batches of B. anthracis Sterne and B. anthracis Ames at 1010
CFU/ml. Because B. 145
anthracis Sterne lacks pXO2 and is exempt from select agent regulations, we utilized the Sterne 146
strain as a surrogate for virulent B. anthracis to develop the procedure prior to validating the 147
results using fully virulent strains. Dose-inactivation of a geographically diverse isolate, B. 148
anthracis Zimbabwe, was also tested. Spore preparations were treated incrementally with ~5 149
kGy increments, and the population of viable spores remaining was determined by serial dilution 150
and plating. Actual radiation doses delivered were monitored by measuring the increase in 151
absorbance of radiochromic film dosimeters that had been attached to each 15 ml conical tube 152
containing the spore aliquots. Dose-inactivation curves from USAMRIID and ECBC are shown 153
in Figure 2. Curves were similar across all batches studied, and were similar between both 154
institutions and among three differing genetic backgrounds. From the slopes of the plots and the 155
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
8
intercepts, D10 and SALs of 10-6
were determined, which are listed in Table 2. We conducted a 156
statistical analysis to determine the average dose for each institute and the 95% confidence 157
intervals for each of the parameters. D10 values for each batch ranged from 1.66 to 2.98 kGy, 158
and DSAL ranged between 29.8 and 46.2 kGy, with the upper bound of the 95% CI of 3.55 kGy 159
and 52.4 kGy for D10 and SAL(10-6
), respectively (Table 3). Our statistical comparison revealed 160
neither significant differences between spore batches nor between institutions (i.e., differences in 161
irradiators and experimental processing), with the exception of a modest but statistically 162
significant difference between the mean DSAL determined for the Ames strain between ECBC 163
and USAMRIID (43.77 versus 37.95 kGy, respectively, p = 0.0089). Overall, averaged over 15 164
inactivation curves covering three different strain backgrounds, the mean D10 was 2.32±0.09 165
kGy, and the mean DSAL was 40.10±1.20 kGy (Table ). 166
167
Analysis of spore integrity/morphology - To determine whether extensive damage to the spore 168
structure had occurred as a result of γ-irradiation, we examined the spores under phase-contrast 169
microscopy. Unirradiated spores were highly refractile (as expected), whereas irradiated Sterne 170
spores had lost much of their refractility (Figure 3). The spore darkening suggests at least partial 171
loss of Ca-dipicolinate and partial rehydration of the spore core. Comparison of varying doses 172
(up to 50 kGy) of unirradiated and irradiated spores by transmission electron microscopy 173
revealed no gross structural damage or morphological alterations (i.e. coat fragmentation or 174
significant damage to the exosporia). 175
At γ-irradiation doses of 15-25 kGy, the morphology of recovered colonies exhibited 176
progressively dramatic variation (Figure 4). While colonies originating from unirradiated spores 177
displayed uniform morphology, spore preparations that had been irradiated with ~15 kGy yielded 178
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
9
heterogeneous colonies in both size and morphology. When spores treated with ~20 kGy were 179
plated, the morphologies were highly variable. Some colonies appeared to expand in a 180
filamentous or “dendritic” pattern, whereas others retained the typical flat, circular, rough 181
morphology with undulate edges. 182
Detection of viable spores in irradiated materials - Since spore recovery at high densities has 183
been shown to be impaired due to the presence of high levels of alanine racemase enzyme (45), 184
we also sought to test whether direct plating yields accurate counts of viable spores, particularly 185
when viable spores are plated along with large numbers of dead spores. We hypothesized in 186
particular that high densities of non-viable spores would inhibit recovery of viable spores due in 187
part to the remaining activity of alanine racemase in the irradiated/dead spores. To test this 188
hypothesis, we spiked irradiated Sterne spores at densities of 1010
CFU/ml with known quantities 189
(~2000 CFU/ml) of viable, unirradiated spores of a strain of B. anthracis Sterne that contains a 190
short genetic tag or “barcode” in the chromosome. Recovery of viable spores by plating was 191
reduced by approximately half in the presence of 109 inactivated spores (Figure 5A). Some of 192
the recovered colonies exhibited a dendritic morphology reminiscent of colonies recovered from 193
near the sterility point (Figure 5A, inset). We then tested whether higher concentrations of dead 194
spores would further inhibit recovery of viable spores. When irradiated spores were 195
concentrated to 1011
CFU/ml prior to spiking (resulting in a mass of 1010
inactivated spores in 196
100 µl samples), recoveries were reduced by 80-95% (Figure 5B). In contrast, recovery of 197
viable spores was not appreciably inhibited when the density of irradiated spores plated with the 198
viable spores was less than 108 (data not shown). All colonies recovered in this experiment 199
(n=88) were shown by barcode-specific PCR to contain the barcode (data not shown). When we 200
pre-treated the viable, barcoded spores with the germinants alanine and inosine (10 mM of each 201
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
10
germinant) prior to adding them to the dead spore mass, near complete recovery of spiked spores 202
was achieved (Figure 5C). However, addition of the germinants after the viable spores were 203
added to the dead spore mass did not promote complete recovery of the viable spores (not 204
shown). 205
Dilution of irradiated spores into liquid culture - We also tested whether dilution of the 206
irradiated spore mass into liquid broth would aid in recovery of viable spores. 100% of 207
irradiated aliquots (one from each spore preparation) were diluted 1:500 (v/v) into three culture 208
flasks containing 500 ml of tryptic soy broth (TSB) and grown in shaking culture at 37 °C for 209
two weeks. The OD600 of the culture decreased within 24 h by approximately 50%, which 210
corresponded to the loss of spore refractility in the irradiation-exposed spores. The OD600 of the 211
suspensions then remained steady at ~0.03 for two weeks, suggesting that no viable spores were 212
detectable in the samples. Furthermore, no viable spores could be cultured from the culture 213
flasks at any point following inoculation. 214
215
Evaluation of effect of post-irradiation incubation on potential spore reactivation - After the 216
unintended shipment of viable spores by the DoD, it was proposed that spores could potentially 217
possess mechanisms to allow for recovery after exposure to radiation, a hypothesis colloquially 218
referred to as the “Zombie Spore Hypothesis” (30). Under this hypothesis, spores “recover” due 219
to DNA repair mechanisms that are active prior to the initiation of the germination program. We 220
tested this hypothesis in several different experiments. Initially, we examined the impact of short 221
term post-irradiation storage at 4 °C and at room temperature. After realizing that multiple 222
institutions have different procedures for viability testing, it became clear that the time between 223
irradiation and viability confirmation could be a confounding variable. In order to address this, 224
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
11
we performed viability testing on samples within 30 min of irradiation and then stored the 225
irradiated material at 4C for long term storage. We re-tested the viability of the irradiated 226
spores at 2 and 4 weeks post-irradiation. No increase in titer was noted for lethally irradiated 227
materials (Figure 6A). As described in Figures 6B and 6C, there was evidence for a slight 228
increase in viability but only at very specific suboptimal doses of radiation (approximately 10-20 229
kGy). The same trends were observed with spores of both Sterne strain (Figure 6B) and the 230
Ames strain (Figure 6C) of B. anthracis. 231
We also examined the impacts of extreme holding times of greater than 1 year. B. anthracis 232
Sterne samples that had been exposed to 35 or 40 kGy and then held for greater than 1 year at 4 233
C were suspended in TSB or brain-heart infusion (BHI) broth to look for any evidence of spore 234
viability. Eight samples were tested at USAMRIID and six samples were tested at ECBC, and 235
there was no evidence of viable B. anthracis in any of the samples (data not shown). Lastly, we 236
examined Sterne spores that were irradiated, frozen at -80 C, and then thawed; again neither 237
USAMRIID nor ECBC observed any restored viability in this scenario (data not shown). 238
The impact of radiation on enzyme activity and DNA integrity – We sought to determine the 239
effects of γ-irradiation on the integrity of known spore components. We reasoned that large 240
macromolecules such as DNA would be more susceptible to degradation than smaller targets 241
such as proteins. We therefore examined the activity of alanine racemase, an important spore 242
enzyme that is localized in both the exosporium and the spore coat, and the integrity of DNA 243
from B. anthracis Sterne spores. The alanine racemase activity was suggested to play a role in 244
the “masking” of viable spores during viability testing. In order to address this concern, we 245
wanted to establish baseline effects of radiation on the alanine racemase activity associated with 246
B. anthracis spores. As presented in Figure 7A, the enzymatic activity associated with spore 247
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
12
preparations was assayed using whole spores that were either boiled (positive control for 248
decrease in enzymatic activity), exposed to 0 kGy, or exposed to 50 kGy. The spores that were 249
exposed to 50 kGy were either irradiated on wet ice or at ambient temperature of the irradiator, 250
representing USAMRIID’s and ECBC’s irradiator configurations, respectively. A dose of 50 251
kGy had a significant effect on the alanine racemase activity associated with B. anthracis Sterne 252
spores irradiated at room temperature (P <0.01) or on wet ice (P = 0.01) compared to untreated 253
spores (0 kGy). There was also a statistically significant difference observed when comparing the 254
alanine racemase activity associated with spores irradiated at ambient temperature compared to 255
spores irradiated on wet ice (P <0.01). The enzymatic activity was determined within 24 h of 256
irradiation and again at 14 days post-irradiation. There was no recovery of alanine racemase 257
activity during this hold time at 4 °C, suggesting that there was no apparent repair of enzyme 258
function associated with time after 14 d at 4 °C. 259
To determine the degree of fragmentation of DNA in irradiated samples, we analyzed 260
genomic DNA isolated from vegetative cells, spores, and irradiated spores. As expected, 261
preparations from spores and vegetative cells yielded DNA of high-molecular weight. DNA 262
from spores also had some lower-molecular weight components. In contrast, DNA from 263
irradiated spores was highly sheared with little high-molecular weight DNA visible (Figure 7B). 264
Effects of γ-irradiation on spore detection assays – We isolated genomic DNA from irradiated 265
and unirradiated spores and performed quantitative qPCR on equal quantities of genomic DNA. 266
No difference in the sensitivity of the assays employed was noted (data not shown). These 267
results were consistent with previous reports (17, 18, 29). Likewise, lateral flow immunoassays 268
specific for B. anthracis were obtained from the Defense Biological Product Assurance Office 269
(DBPAO). No differences in limit of detection of these assays was noted when irradiated spores 270
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
13
were utilized, although the intensity of the positive signal was reduced when irradiated spores 271
were applied to the test strips of the lateral flow assay. 272
Discussion 273
In this work we present an integrated, end-to-end procedure for the standardized production of 274
irradiated spores of B. anthracis. We were able to apply the quantitative concept of the Sterility 275
Assurance Level to the residual risk of recovering a viable spore in preparations of irradiated 276
spores. 277
The spore preparation method described here yields a high concentration (approximately 1010
278
CFU/ml) of viable spores in a monodisperse suspension of uniformly sized particles. It is based 279
on previous work in our laboratories with B. thuringiensis Al Hakam and B. anthracis ΔSterne 280
(41, 43, 44, 46, 47). Including 0.1% Tween-80 in our suspension buffer and all diluent buffers to 281
reduce aggregation minimizes the effects of clumping on downstream analyses and reduces any 282
protective effects that aggregation may have during exposure to γ-radiation. Spores of 283
exosporium-containing Bacillus spp. tend to agglomerate and stick to surfaces (34) due to spore 284
hydrophobicity (35-39) which can be specifically attributed to the exosporium. Species lacking 285
exosporia, such as Bacillus subtilis or B. atrophaeus (40, 41), are considerably less prone to 286
aggregation. produces consistent results, as shown by the ability not only to produce three 287
virtually identical batches using multiple shake flasks per batch, but to do so at three different 288
institutions with comparable results in three different genetic backgrounds. 289
Likewise, the irradiation procedure described here produced almost superimposable results in 290
two different institutions with two differently configured irradiators. USAMRIID’s irradiator is 291
configured such that multiple 60
Co source elements surround the sample, producing a uniform 292
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
14
field of radiation, whereas ECBC’s irradiator has three independently operable sources at the rear 293
of an approximately cubic chamber, producing a field that attenuates with increasing distance 294
from the source. In addition, USAMRIID’s irradiator configuration permitted the use of ice to 295
keep the sample cold, whereas this was not possible in ECBC’s irradiator due to the size 296
limitation of the chamber. In both cases, D10 and DSAL values extrapolated from the inactivation 297
curves were nearly identical, although the differences in irradiator configuration may contribute 298
to some of the higher variability observed in the ECBC runs. By utilizing independent biological 299
replicates, we also accounted for batch-to-batch variability as a potential contributor to 300
differences in radiation sensitivity, provided that conditions for production of the initial spore 301
material are kept consistent. 302
Unlike previous reports (15, 17, 18, 26, 33), our method incorporates the known inhibitory 303
effects on germination of plating spores at high spore densities into our standard procedure for 304
quantitating residual viable spores (45). This experiment was motivated by our findings that 305
viable spores were not recovered at doses of at least 30 kGy, whereas the material distributed 306
prior to 2015 by DoD had been irradiated with considerably higher doses, yet was shown to 307
contain low concentrations of viable B. anthracis spores (30). It is unlikely that the absence of 308
detectable viable spores in our study is due to complete inhibition of germination of remaining 309
viable spores, since the dead spore mass plated in the generation of the kill curves only 310
marginally inhibited recovery of viable spores from deliberately spiked samples. At the upper 311
end of the dose range utilized in the kill curves, we would have expected only ~50% inhibition 312
when 100 µl of the inactivated material was plated (Figure 5A). This inhibition of germination 313
could be due to spatial contraints limiting contact of viable spores with the TSB or more likely 314
due to residual alanine racemase activity associated with the dead spore mass. Plating spores at 315
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
15
higher densities than those we routinely utilized in this study (e.g. 1011
CFU/ml, such as those 316
formerly produced by the DoD (30)) resulted in 80-95% inhibition of recovery of the barcoded, 317
spiked spores (Figure 5B). It is theoretically possible that residual viable spores in the material 318
formerly produced by DoD may have escaped initial detection due to inhibition of germination 319
and/or outgrowth by the dense mass of dead spores. However, the differences between our 320
conditions and those used to grow, prepare, and irradiate the material formerly distributed by 321
DoD make direct comparison of the two scenarios impossible. Our results indicate that, due to 322
the inhibitory effect of the dead spore mass, approximately 20% of the undiluted inactivated 323
material would need to be plated to achieve the equivalent of the suggested 10% sample for 324
determining non-viability (48). Alternately, our results indicate that diluting the inactivated 325
spores by a factor of 10 prior to plating for viability generally eliminates the inhibitory effect of 326
the dead spore mass. 327
It is also unlikely that B. anthracis spores irradiated to a SAL of 10-6
can recover from radiation 328
damage after extended periods of storage before plating. Our DNA profiles show extreme 329
degrees of fragmentation (Figure 7), a type of damage that is typically repaired by non-330
homologous end-joining (NHEJ) in irradiated spores of Bacillus subtilis. NHEJ is thought to 331
occur during germination with the resumption of metabolic activity (49, 50), and would not be 332
active in ungerminated spores. When viable spores can be detected immediately post-irradiation, 333
the number of recoverable CFUs from stored (“held”) samples increases only marginally within 334
approximately 1 week of irradiation. However, it is not clear whether this observation is due to 335
actual germination and proliferation of bacilli within the sample or whether the increase in 336
recoverable CFUs represents the ongoing disaggregation of clusters of spores that may have 337
protected a minuscule fraction of the spores from irradiation. We consider the latter possibility 338
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
16
unlikely due to the monodisperse nature of our spore suspensions, but we cannot formally 339
exclude the possibility that a small number of spores are present in clusters that are not readily 340
found in the microscopic surveys performed here. In contrast, when viable spores were not 341
detected immediately post-irradiation, even extended hold times of up to 4 weeks at room 342
temperature or at 4° C, or over a year at 4° C, did not promote significant recovery of viable 343
spores from these materials. We therefore believe that, under the conditions described here, 344
irradiation to an SAL of 10-6
is sufficient to render a 3 ml suspension containing 3 x 1010
spores
345
inactive and non-viable. 346
Because the spore preparation described here is intended for use across the Chemical and 347
Biological Defense Enterprise as a standard reagent to test DoD-fielded and candidate detection 348
systems, it was necessary that the requisite properties of the spore be preserved through the 349
irradiation process. The damage to spores in the irradiation experiments likely results from a 350
combination of direct radiation and of oxidative and thermal damage, the latter of which may be 351
mitigated by maintaining the spores on ice during the procedure (Figure 7B). We have 352
documented several altered colony morphologies associated with apparent -radiation damage 353
(Figure 4). The “dendritic” morphology may be due in part to effects of the mass of dead spores, 354
as colonies recovered from unirradiated, barcoded spores exhibited a dendritic morphotype when 355
plated on high densities on dead spores. However, the small colony morphotypes likely arise 356
from irradiation-induced damage. While the exact causes of the phenotypic changes are 357
unknown, their observation is important as an indication that the dose of -radiation delivered is 358
clearly sub-optimal but likely approaching the lethal dose which would result in high confidence 359
SALs. Based on our results with a standard hand-held lateral-flow immunoassay, sensitivity is 360
not substantially compromised when irradiated material is applied to the assay tickets, nor is the 361
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
17
utility of the material diminished for detection using qPCR assays, in accordance with previous 362
work (17, 18, 29). We cannot exclude the possibility that spores may be damaged in ways not 363
accounted for in this study, particularly with respect to membrane and coat components. Of 364
particular concern may be the integrity of the polymeric components of the spore coat and the 365
structure of the exosporium. Notably, the spore germ cell wall and cortex consist of high-366
molecular weight peptidoglycan components that would form large macromolecular targets for 367
radiation or free-radical damage (3, 9, 51, 52). These larger molecular complexes would likely 368
be more sensitive to radiation than individual proteins, since radiation sensitivity is a function of 369
molecular target size (53). Our previous work indicated that irradiated B. atrophaeus spores lose 370
the ability to bind malachite green spore stain (29), suggesting that irradiation can impact the 371
integrity of the spore coat, and the loss of refractility observed in this study suggests that 372
irradiated spores may have suffered similar damage to one or more components of the spore 373
envelope . 374
We experienced a single instance (out of a total of 65 samples irradiated at >30 kGy) in which 375
viable spores were recovered after irradiation. Analysis of the procedure used to dilute and plate 376
for viable spores in that experiment revealed that the samples had been diluted and plated 377
beginning with the un-irradiated materials and concluding with the highest dose points. 378
Subsequently, we ensured that the samples were plated in order of increasing expected titer of 379
viable spores, and no viable organisms were detected in the high-dose samples from subsequent 380
irradiation experiments. Given the high titers of viable spores in these samples, cross-381
contamination of inactivated samples is possible — a 1 nl droplet of spore materials prepared as 382
described here would contain 104
viable spores, a level which would contaminate a 3 ml aliquot 383
of inactivated spores to a level of 3 × 103 CFU/ml. The latter titer is well within the range 384
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
18
observed in the samples analyzed for the DoD report (reference (30) and S. Sozhamannan, 385
personal communication). Based on the consistency of our dose-inactivation curves and the 386
number of samples successfully inactivated in this study, we believe that previous “irradiation 387
failures” (26, 30) may actually have been the result of inadvertent contamination with extremely 388
small volumes of high-titer spore material. Therefore, principles of aseptic technique must be 389
practiced stringently, with particular attention to any potential exposure of irradiated spore 390
suspensions to materials or surfaces (e.g. pipettors) containing viable spores. At a minimum, 391
irradiated materials should be manipulated in a rigorously cleaned biosafety cabinet in which no 392
viable spores have been handled prior to the experiment. To further reduce the risk of cross-393
contamination of material destined for removal from containment facilities, a dedicated set of 394
pipettors should be utilized for irradiated material that should be distinct from any pipettor used 395
for preparing or characterizing the unirradiated spore preparation. Finally, we recommend that 396
both the irradiated material itself and any cultures used to check for viability be transferred from 397
the biosafety cabinet prior to any manipulation of viable spores. 398
We believe that it is critical to note that the irradiation procedure presented here is applicable 399
only to those conditions described in this study. Significant deviations from the procedure 400
described here (e.g. changes in spore concentration, volume of spore suspension, media or 401
diluent composition, irradiation dose rate, etc.) could potentially result in unpredicted effects on 402
radiation sensitivity. Indeed, resistance of B. subtilis spores to UV, wet heat, and hydrogen 403
peroxide can be altered by changing the growth conditions (54, 55). Thus, it is recommended 404
that, prior to producing irradiated spore material in a new laboratory setting or following changes 405
to the spore preparation methods, D10 and DSAL values determined in this work be independently 406
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
19
verified, and that 100% sampling of the initial batches of produced material be conducted to 407
ascertain that no viable spores escape either irradiation or detection. 408
Materials and methods 409
Bacillus anthracis strains - The Sterne strain (34F2) was obtained from the Unified Culture 410
Collection at USAMRIID (Frederick, MD): Unified Culture Collection Identifier AGD 0000844 411
BACI012. The parent strain for construction of a barcoded Sterne variant was obtained from S. 412
Leppla (National Institutes of Allergy and Infectious Diseases, Bethesda, MD). The Ames strain 413
utilized in this study was obtained from the USAMRIID collection or the Lovelace Research 414
Institute. The Zimbabwe strain utilized in this study was obtained from the USAMRIID culture 415
collection. Strains were routinely cultured on tryptic soy agar (TSA, Difco), TSA containing 5% 416
sheep’s blood (TSAB, Remel), or on brain heart infusion (BHI) Agar (Difco). Spectinomycin 417
and kanamycin were utilized (when required) at 250 and 20 µg/ml, respectively. 418
Strain construction – Barcode sequence and oligonucleotide primers used for construction of the 419
barcoded B. anthracis Sterne strain are listed in Table 5. Btk-Barcode1 is a 148 bp unique DNA 420
sequence designed in silico using the Barcoder algorithm (Lux et al., manuscript in preparation) 421
and synthesized by Integrated DNA Technologies Inc. A neutral chromosomal location for 422
barcode insertion, between the convergently transcribed genes BAS2302 and BAS2303 of the B. 423
anthracis Sterne chromosome (RefSeq accession number NC_005945.1), was selected using the 424
TargetFinder algorithm (details to be published elsewhere). We adopted selection rules from (56) 425
in combination with the PATRIC database (57) and RNA-seq data (58, 59) to ensure the absence 426
of annotations and transcription within the region. Approximately 500 bp flanking each side of 427
the barcode insertion point were separately PCR amplified from the B. anthracis Sterne 34F2 428
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
20
chromosome. Btk-Barcode1 was PCR amplified from the synthesized plasmid (pSMART-Btk1). 429
The three amplicons were joined by overlap extension PCR (60) using primers Ba1_UpFlank_F 430
and Ba1_DownFlank_R (Table 5) and mediated by the overlapping DNA ends generated during 431
the initial PCR step. The resulting DNA fragment contained Btk-Barcode1 flanked on both 432
sides by B. anthracis DNA. Following gel extraction (QIAquick Gel Extraction Kit, QIAGEN), 433
the DNA fragment was cloned into plasmid pRP1028 (61) by digestion with the restriction 434
enzymes BamHI-HF and KpnI-HF (New England BioLabs Inc.) and ligation. This pRP1028-435
derivative was introduced into B. anthracis Sterne 34F2 and Btk1-Barcode1 was incorporating 436
into the chromosome using the markerless allelic exchange strategy described previously (61). 437
Btk-Barcode1 insertion at the desired site was confirmed by PCR amplification and sequencing. 438
Whole-genome sequencing (MiSeq, Illumina) was also performed to verify the absence of 439
substantial off-target mutations. When used to spike irradiated materials, barcoded strains were 440
prepared and titered according to the procedure described below, diluted in PBS/0.1% Tween 80 441
and added to irradiated spore suspensions to a final concentration of ~2000 CFU/ml. In some 442
experiments the barcoded spores were exposed to L-alanine and inosine (10 mM) prior to being 443
spiked into the dead spore mass. In other experiments, L-alanine (100 mM final concentration) 444
and inosine (6.7 mM final concentration) were added to the dead spore mass before the latter was 445
spiked with the barcoded spores. 446
Preparation of B. anthracis spores – Spores were prepared in broth and characterized as 447
previously published (41, 47, 62, 63). Sporulation medium was 2.5% nutrient broth (NB) 448
amended with CCY salts (41, 62, 64, 65) at pH 7.0, as described previously (46). A 2.63% 449
solution of nutrient broth and 30x KPO4 buffer (pH 7.0) (CCY buffer) were sterilized as 450
independent components. CCY divalent cations were sterile-filtered and stored at -80°C. 451
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
21
Nutrient broth and CCY buffer were combined before addition of CCY divalent cations to 452
mitigate divalent cation-phosphate precipitation. TSA was streaked with primary glycerol stocks 453
of B. anthracis that were frozen in long-term storage at -80 °C. After incubation for 16±2 h at 37 454
°C, a single colony from a TSA plate was transferred to 10 ml of sporulation medium (pre-heated 455
to 37 °C) and vortexed for 30 s. Pre-aerated and pre-heated sporulation medium (200 ml medium 456
in 1-1 baffled flasks with filter caps; Corning #431403) was inoculated with 0.6 ml from the 10 457
ml of inoculum. The Erlenmeyer flasks were then incubated at 34 °C with shaking (300 rev min-
458
1) for 72 ± 2 h in a New Brunswick Scientific shaker/incubator. Sporulated cultures were 459
amended with 35.5 ml of 20% Tween-80 (final concentration 3%) and incubated an additional 24 460
± 2 h, 34 °C at 300 rev min-1
to disperse (“unclump”) spores. Spores were harvested by 461
centrifugation at 2,000 × g, 20 °C for 10 min. Spores were washed twice with 200 ml of 3% 462
Tween-80 at room temperature (22±4 °C) for 24±2 h at 200 rev min-1
. Spores were resuspended 463
in 10-20 ml of 0.1% Tween-80 as previously published (34, 41-44) to a final concentration of ~1 464
× 1010
CFU/ml, aliquoted into 3 ml portions, and then characterized via light microscopy, and 465
BCM analysis (47, 62, 63). Titers of heat-resistant spores were obtained by heating spore 466
preparations to 65 °C for 30 min and plating serial dilutions of untreated versus heat-treated 467
spore preparations. BCM analysis was used to assess spore clumping, determine spore size, and 468
quantify spore cleanliness. TSAB were used to verify that the starting strains and subsequent 469
isolates were non-β-hemolytic. B. anthracis Ames strain spores were made as described above, 470
with the exception being the Ames strain and Zimbabwe strain spores produced at USAMRIID 471
were incubated with shaking at 200 rev min-1
. 472
Irradiation of spores – Three ml aliquots of spores in 15 ml conical tubes were packaged either 473
into a Safe-T-Pak STP-100 Category A Reusable Shipping System (Saf-T-Pak, Inc., Hanover, 474
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
22
MD) at ECBC or into triple sealed plastics bags at USAMRIID and irradiated in a JL Shepherd 475
irradiator (ECBC: Model 484; USAMRIID: Model 109-68). The differences in irradiation 476
procedures at this stage arose because spore preparation and irradiation at ECBC were carried 477
out in separate buildings, requiring transport of Select Agent materials in appropriate containers 478
between facilities. In most cases, USAMRIID maintained samples on ice during irradiation, 479
whereas temperature of the samples could not be regulated at ECBC due to the space constraints 480
of the irradiator. Irradiation times to achieve the desired dose were determined by the position of 481
the sample in the irradiator and extrapolating the administered dose based on the age of the 482
respective cobalt sources. 483
Radiation dosimetry – In most experiments presented here, B3 WINdose radiochromic film 484
dosimeters (GEX Corporation, Centennial, Colorado, USA) were applied to the outside of the 15 485
ml conical tubes. Following irradiation, dosimeters were heat-fixed at 60 °C for 15 min. Films 486
were read at 525 nm on a spectrophotometer (Genesys 20, Thermo Scientific) containing a 487
dosimeter holder. Using a reference table provided by GEX, the absorbance value was translated 488
to irradiation dose received. 489
Electron microscopy - Standard methods for transmission electron microscopy (EM) were 490
employed. B. anthracis spore samples receiving varying doses of irradiation were fixed with 1% 491
glutaraldehyde and 4% formaldehyde in 0.1M phosphate buffer. Post fixation was performed for 492
1 h at room temperature in phosphate buffer containing 1% osmium tetroxide and contrasted in 493
ethanolic uranyl acetate before dehydration in a graded series of ethanol rinses and propylene 494
oxide. The samples were embedded into EMbed-812 embedding medium (Electron Microscopy 495
Sciences; Hatfield, PA) overnight at room temperature, and the samples were then sectioned into 496
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
23
90-nm sections. These sections were counterstained with uranyl and lead salts. All EM samples 497
were examined using a JEOL 1011 transmission electron microscope. 498
Detection and enumeration of viable spores – Following irradiation, spore suspensions were 499
serially diluted 1:10 (v/v) into PBS containing 0.1% Tween-80, briefly vortexed, and plated in 500
triplicate (100 µl) onto TSAB. Plates were incubated at 37 °C and plates with well-separated 501
colonies were enumerated after 24 and 48 hr. Plates with no growth were incubated for up to 30 502
days and monitored for the appearance of colonies. For each lot of spores, the log10 of the 503
concentration of viable spores at 48 h incubation was plotted versus the dose of irradiation 504
received to produce a kill curve. The D10 value, the dose that kills 90% of the spore population, 505
was calculated as the negative reciprocal of the slope following linear regression analysis. The 506
irradiation dose to achieve a SAL of 10-6
was extrapolated from the regression plot. 507
In some experiments, viable spores of a genetically-tagged strain of B. anthracis Sterne (see 508
above) were spiked into the irradiated spore suspension to a final concentration of ~2000 509
CFU/ml. In spiking experiments, 10 µl of barcoded spores at 2 × 104
CFU/ml in PBS/0.1% 510
Tween-80 were added to 100 µl of irradiated spore suspension or, as a control, to 100 µl of 511
diluent buffer. The spiked samples were held at room temperature for 4 hours before being 512
plated onto TSAB. Plates were enumerated following overnight incubation at 37 °C. 513
Three independent batches of irradiated B. anthracis Sterne spores were evaluated for viability 514
using 100 percent sampling of one (3 ml) aliquot irradiated at 40 kGy. The aliquot was diluted 515
to a volume of 15 ml in tryptic soy broth (TSB) and split evenly into three 2 l non-baffled 516
disposable sterile flasks with filtered caps, each with 500 ml TSB, to obtain cultures with optical 517
densities at 600nm (OD600) of approximately 0.05. The cultures were incubated at 37 °C with 518
shaking at 225 rpm for two weeks. The OD600 was monitored at 0, 1, 3, 7, and 14 days post-519
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
24
inoculation. Microscopic morphology under 100x magnification was monitored at day 0, 1, and 520
14, and viability was assessed by plating on solid medium on days 7 and 14. For viability 521
assessment by plating, one ml of culture was centrifuged for 10 min at 3000 x g, 900 µl of 522
supernatant was removed, and the remaining 100 µl was used to resuspend the spores prior to 523
plating the entirety on sheep TSAB plates. The plates were incubated at 37 °C and evaluated for 524
1 week to ensure non-viability of the spores. 525
526
Evaluation of post-irradiation hold time and temperature on viability assessment - Four tubes 527
from each of the three NSWC batches of B. anthracis Sterne were prepared for irradiation at 528
either ECBC or USAMRIID as described above. The turntable of the irradiator at ECBC was 529
non-functional; therefore, the container was rotated manually by 90° at 5 kGy intervals. One 530
tube from each batch was removed following irradiation with nominal doses of 15, 20, 25, and 531
40 kGy. Actual doses for each experiment were verified by dosimetry as described above. In 532
some experiments, the samples were divided following irradiation. Half of the material was held 533
at room temperature, and the other half of the material was held at 4 °C. All of the material was 534
plated in triplicate on TSAB plates at several weeks post irradiation (hold time is indicated in 535
each figure). All plates were incubated at 37 °C and were counted 24 and 48 h after plating. 536
Additionally, irradiated Sterne spores were maintained at 4 °C for over 1 year. 537
Evaluation of freeze-thaw cycle effects on viability assessment - B. anthracis samples that were 538
used to generate the kill curve data were used to evaluate the effect of a freeze/thaw cycle on 539
irradiated B. anthracis spores. 300 µl aliquots of ~40 kGy irradiated spores were placed at -80 540
°C overnight. The next day the samples were thawed at room temperature, plated in triplicate on 541
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
25
TSAB plates (100µl per plate), and incubated at 37 °C. Plates were observed at 24 and 48 h for 542
growth. 543
Alanine racemase activity assay – Assays were performed to measure alanine racemase on the 544
surface of B. anthracis spores (66, 67) . Two individual preparations of B. anthracis Sterne 545
spores were diluted to a working stock concentration of 1 × 108 CFU/ml. Each diluted spore 546
stock was aliquoted into twelve separate screw cap tubes. Four tubes of each stock were boiled 547
for 1 h. Four separate tubes of each stock were irradiated at 50 kGy, on either wet ice or at room 548
temperature, and the remaining aliquots were untreated and stored at 4 °C. Boiled control 549
samples were boiled the same day on which the experimental samples were irradiated. After the 550
sample preparation, a reaction mixture was prepared with the following final concentrations of 551
reagents in water for injection: 10 mM tricine pH 8.5, 10 mM β-nicotinamide adenine 552
dinucleotide sodium salt (β-NAD), 0.15 U/ml L-alanine dehydrogenase, and 0.1 mM D-alanine. 553
This reaction mixture was made fresh for every assay and kept on ice in dark conditions before 554
use. A 90 µl aliquot of the reaction mixture was added to the experimental wells in a Costar 555
black/clear bottom microtiter plate. A 120 µl volume of reaction mixture was added to the plate 556
as a medium control and 120 µl of Tris buffer (50mM Tris, pH 8.0) was added to separate 557
background wells. Spores were dispensed into a separate Falcon 3007 clear U-bottom plate to 558
prevent the reaction from starting until all media had been added to the appropriate wells. The 559
spores (30 µl) were then transferred to the experimental wells. After 15 min at room temperature, 560
the plate was inserted into a SpectraMax i3x Multimode Detection Platform. The fluorescence 561
was measured every 20 min for 1 h (Ex 340/ Em 460). 562
563
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
26
Statistical analysis - For each trial, the relationship between radiation dose (kGy) and colony 564
count (CFU) was estimated under the generalized-linear regression model having a structural 565
component 566
𝑙𝑜𝑔( 𝐴𝑣𝑒𝑟𝑎𝑔𝑒(𝐶𝑜𝑙𝑜𝑛𝑦 𝐶𝑜𝑢𝑛𝑡) ) = ∝ + 𝛽 ∗ 𝐷𝑜𝑠𝑒 + log ( 𝑉𝑜𝑙𝑢𝑚𝑒 ∗ 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛)
with errors following a negative binomial distribution. The free parameters ∝ and 𝛽 were 567
estimated from the data, and represent the intercept and slope of the log-linear model, 568
respectively. This model is equivalent to the standard single-target single-hit model as discussed 569
by Bodgi et al (68). The dose required for each 10 fold reduction in the colony count (D10) is 570
estimated as log (0.1)
𝛽. The dose required to achieve an expected colony count of 10
-6 is estimated 571
as log( 10−6) − 𝛼
𝛽 . Confidence intervals are based on large sample delta-method approximation. 572
Analysis was implemented in SAS®
PROC NLMIXED SAS3 version 9.4. A few outliers were 573
removed prior to model fitting. In the NSWC/20170620 experiment, the 15.4 kGy dose level, 574
dilutions -6 and -7 were removed along with a single observation labeled as likely hemolysis. 575
These were technical errors, marked as such in the data. In the ECBC2/20170705 colonies were 576
observed in a single aliquot that had been irradiated with 44.4 kGy. As these were likely due to 577
contamination with unirradiated spores (see Results and Discussion, above), these data were not 578
included in the fit and were marked as a technical error in the source data files. Several of the 579
experiments showed a pronounced shouldering effect at low doses, wherein the dose response 580
did not take effect at doses near 0 kGy. To improve the overall model fit at higher dose levels, 581
the 0 kGy points were not considered for the purpose of model fitting. The statistical 582
significances of strain and institute effects were obtained by entering the unweighted estimates of 583
D10 and DSAL into Welch’s t-test. No adjustment was applied for multiple comparisons. The 584
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
27
alanine racemase assays were processed as follows: For each preparation, plate blanks were 585
subtracted, technical replicates were averaged, and the maximum linear rate (RFU per min.) was 586
estimated by ordinary least squares linear regression. Rates were compared between treatment 587
groups by an ANOVA model, controlling for the effect of spore stock. Arithmetic mean rates 588
are reported as ratios to boiled control, with confidence intervals obtained by standard delta 589
method approximations to the variance of the ratio of statistically independent arithmetic means. 590
Analysis is implemented in SAS version 9.4 SAS PROC GLIMMIX. 591
592
593
Table 1: Specifications for standarized spore preparation for B. anthracis strains and surrogates 594
Property Requirement Method
Titer 108 heat-resistant CFU/ml in initial
sporulation medium; 1010 CFU/ml in
final preparation
Heat-shock 107 spores for 30 min
at 65 C, quantitate by serial
dilution plating on TSA medium
Size 1.0 – 1.5 µm volume-equivalent
spherical diameter Measure 500 spores using a
BCM
Purity 95% pure spores with minimal
vegetative cell debris
Examine 100 particles per spore
preparation by light microscopy.
Spores are bright, refractive
particles.
Clumping or
aggregation
Unclumped, individual spores Prepare and dilute spores in
buffer containing 0.1% Tween-
80. Examine 100 spores by light
microscopy and 500 spores by
particle analysis via BCM
595
596
Table 2: Results of individual dose-inactivation experiments 597
Strain Irradiator Spore Preparation/Irradiation
Date
D10 (95% CI)
kGy
DSAL (95% CI)
kGy
AMES ECBCc NSWC/20170620 2.35 ( 2.03 , 2.67 ) 43.86 ( 40.23 , 47.48 )
ECBC1/20170627 2.02 ( 1.10 , 2.95 ) 41.53 ( 30.19 , 52.86 )
ECBC2/20170705 2.33 ( 2.22 , 2.45 ) 44.05 ( 42.66 , 45.44 )
ECBC3/20170906 2.46 ( 2.23 , 2.70 ) 45.65 ( 42.89 , 48.41 )
USAMRIID USAMRIID1/20170419 2.19 ( 2.18 , 2.20 ) 37.79 ( 37.55 , 38.04 )
USAMRIID2/20170524 2.04 ( 1.92 , 2.16 ) 36.32 ( 34.93 , 37.72 )
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
28
NSWC/20170621 2.28 ( 2.12 , 2.45 ) 39.75 ( 37.96 , 41.53 )
STERNE ECBCc NSWC1/20160513 2.75 ( 2.48 , 3.01 ) 42.59 ( 39.67 , 45.50 )
NSWC1/20160416a 5.11 ( 4.29 , 5.94 ) --
NSWC2/20160517 2.20 ( 1.95 , 2.45 ) 37.02 ( 34.06 , 39.98 )
NSWC3/20170519 1.66 ( 1.23 , 2.10 ) 29.83 ( 25.18 , 34.48 )
USAMRIID NSWC1/20160422a,b 2.81 ( 2.63 , 2.99 ) 46.22 ( 44.38 , 48.06 )
NSWC2/20160429a,b 2.25 ( 2.16 , 2.34 ) 37.24 ( 36.29 , 38.19 )
NSWC3/20160512 2.98 ( 2.76 , 3.19 ) 44.45 ( 42.27 , 46.64 )
NSWC1/20160607c 2.08 ( 1.99 , 2.17 ) 34.30 ( 33.37 , 35.22 )
ZIMBABWE USAMRIID USAMRIIDZimbabwe20170705 2.45 ( 2.39 , 2.51 ) 40.86 ( 40.17 , 41.56 )
D10: Decimal reduction value. Dose required for 10 fold reduction. 598 DSAL: Sterility assurance level. Dose required for rate per mLl of 10-6. 599 -- Indicates statistics not estimable from the observed data. 600 CI: Confidence Interval. 601 a Tween-80 not present in diluent buffer 602 b Dosimetry not utilized 603 c No ice utilized during irradiation 604 605
606
607 608
Table 3: Inter-laboratory comparison dose-inactivation experiments 609
kGy - Ames kGy - Sterne Difference (P-Value)
D10 ECBC/Dahlgren 2.29 ( 1.99 , 2.59 ) 2.20 ( 0.86 , 3.55 ) 0.09 (P = 0.8089)
USAMRIID 2.17 ( 1.87 , 2.48 ) 2.53 ( 1.84 , 3.21 ) -0.36 (P = 0.1967)
Difference (P-Value) 0.12 (P = 0.3511) -0.32 (P = 0.4441)
DSAL ECBC/Dahlgren 43.77 ( 41.07 , 46.48 ) 36.48 ( 20.59 , 52.37 ) 7.29 (P = 0.1818)
USAMRIID 37.95 ( 33.69 , 42.22 ) 40.55 ( 31.49 , 49.62 ) -2.60 (P = 0.4415)
Difference (P-Value) 5.82 (P = 0.0089) -4.07 (P = 0.4303)
Excluding experiment NSWC1/20160416 610 Values indicate mean (95% CI). 611 P-Values indicate result of Welch’s T-test. 612
613
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
29
614
615
Table 4: D10 and DSAL Overall Summary 616
Statistic N
Mean
(kGy)
SEM
(kGy)
Std Dev
(kGy)
D10 15 2.32 0.09 0.34
DSAL 15 40.10 1.20 4.65
Excluding experiment NSWC1/20160416 . 617
618
Table 5: Oligonucleotide sequences utilized in this study 619
620
621
Primer or
sequence name
Sequence (5ʹ to 3ʹ) a Use
Ba1_UpFlank_F CTGAGGATCCGATGCACA
ACTGCCATCC
Amplification of ~500 bp fragment upstream of barcode
insertion point and amplification during overlap extension
PCR
Ba1_UpFlank_R GACAGAGCTCCTTATCTG
CTTTCTCTGTATATTTTTTAA
G
Amplification of ~500 bp fragment upstream of barcode
insertion point
Ba1_Barcode1_F GCAGATAAGGAGCTCTGT
CTCCAACTC
Amplification of Btk-Barcode1
Ba1_Barcode1_R GGTTCTTCAGCTAGCGGTT
TGAGGCAG
Amplification of Btk-Barcode1
Ba1_DownFlank_F CCGCTAGCTGAAGAACCTA
CCTGCTTC
Amplification of ~500 bp fragment downstream of barcode
insertion point
Ba1_DownFlank_R CGTGGTACCGTTGAAGTA
ATTGGTACAG
Amplification of ~500 bp fragment downstream of barcode
insertion point and amplification during overlap extension
PCR
Ba1_UpOut CGTACTAGCCCCTCTGATG
ATG
Amplification of entire barcode insertion region/sequencing
Ba1_DownOut ACCGCAGTCCGTTCTAATA
TCA
Amplification of entire barcode insertion region/sequencing
Btk-Barcode1 GAGCTCTGTCTCCAACTC
CCAGTATCTTATTTTCTTA
ATAGATTATATCAAGACT
ATTAATTTTACCCGACGC
AGGACCCCACTAAGTGAT
TTCAATATTTAGAAATTA
TTGTAAAATATCTTAACT
TCGCTGCCTCAAACCGCT
AGC
Barcode sequence. Barcode is flanked by SacI and NheI
restriction sites (bold) and was obtained in plasmid
pSMART-Btk1.
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
31
623
Figure Legends 624
Figure 1: Standardized spore preparation of B. anthracis Sterne. Particle size distribution 625
analysis of three independent spore preparations prepared according to methods described in this 626
work. Inset shows monodisperse, phase-bright spores visualized by phase-contrast microscopy. 627
Figure 2: Summary of dose-inactivation curves from 16 independent inactivation experiments 628
performed at A) ECBC and B) USAMRIID. Except as noted, experiments performed at 629
USAMRIID maintained spore suspensions on ice during irradiation. Shaded area represents the 630
95% confidence interval. aDiluent was PBS with no Tween-80.
bIce omitted. 631
Figure 3: Spore ultrastructure analysis. Irradiated and unirradiated B. anthracis Sterne spores 632
were visualized by transmission electron microscopy and phase-contrast microscopy (50 and 40 633
kGy doses, respectively). Bars in electron microscopy represent 500 nm. Phase-contrast images 634
were taken using an oil immersion objective at 1000x magnification. 635
Figure 4: Abnormal morphologies of B. anthracis Sterne colonies recovered from samples 636
irradiated with near-sterilizing doses of 15 and 20 kGy (nominal dose) of γ-radiation. 637
Unirradiated and irradiated spores were diluted to yieldcountable numbers of colonies upon 638
plating. Experiment shown is representative of three independent biological replicates irradiated 639
and plated at two different institutions. Black arrows represent small colony morphotypes; white 640
arrows represent “dendritic” morphotype. 641
Figure 5: Recovery of viable spores is inhibited by the presence of high densities of dead spores. 642
A) Viable spores were spiked to a concentration of ~2000 CFU/ml into 100 µl of irradiated 643
spores at 1010
CFU/ml or diluent buffer and plated onto TSAB. Inset: abnormal morphology of 644
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
32
recovered colonies. B) Effects of concentrating dead spores to 1011
CFU/ml on viable spore 645
recovery. 100 ul of each spore suspension was spiked with 2000 CFU/ml of barcoded B. 646
anthracis Sterne spores and plated onto TSAB. C) Effects of germinants and hold time on spore 647
recovery. Alanine and inosine (AI) were added (10 mM each) to viable spores prior to spiking 648
into slurry of dead spores. PBS was added in lieu of alanine and inosine as a control. Results are 649
the average of two independent experiments. Error bars represent standard deviation. Addition 650
of AI to dead spore mass prior to addition of live spores did not promote recovery of viable 651
spores (not shown) 652
Figure 6: Effects of extended hold times on recovery of spores in irradiated samples of B. 653
anthracis Sterne and Ames spores. A) Representative absolute CFU titers of Sterne spores after 654
post-irradiation holds of 1-4 weeks at 4 °C. B) Increase in viable counts in comparison to t0 655
initial counts performed immediately after exposure to radiation (fold increase). Post-irradiation 656
storage of Sterne spores for 2-4 weeks at 4 °C leads to a relative increase in viable spore titer. A 657
modest increase in recovered viable CFU was observed in some cases when sub-optimal doses of 658
radiation were administered (i.e. 10-20 kGy). C. The same effects were observed for spores of 659
the fully-virulent Ames strain. In cases where there was no detectable growth, a value of 1 660
CFU/ml was used to calculate the respective fold increase, as that was the limit of detection of 661
the plating assay. 662
Figure 7: Spore irradiation affects DNA and enzymatic integrity. A) Alanine racemase activity 663
in unirradiated spores (0 kGy), spores irradiated at room temperature (RT), and spores irradiated 664
on wet ice (ICE). The data depict relative enzymatic activities compared to boiled spores. Two 665
individual spore preparations were used to calculate enzymatic activities. For each test-treatment 666
(radiation on ice or ambient temperature) N=4 or for the unirradiated-0 kGy samples N=8 and 667
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
33
the bars depict 95% confidence intervals (CI). A dose of 50 kGy had a significant effect on the 668
alanine racemase activity associated with B. anthracis Sterne spores irradiated at room 669
temperature (P <0.01) or on wet ice (P = 0.01) compared to untreated spores (0 kGy). There was 670
also a statistically significant difference observed when comparing the alanine racemase activity 671
associated with spores irradiated at ambient temperature compared to spores irradiated on wet ice 672
(P <0.01). The enzymatic activity was determined within 24 h of irradiation and again at 14 days 673
post-irradiation; there was no recovery of alanine racemase activity during this hold time at 4 °C. 674
B) Agarose gel electrophoresis of genomic DNA isolated from vegetative bacilli (Veget.), 675
unirradiated spores, and irradiated spores. Genomic DNA isolated from B. thuringiensis 676
vegetative cells is also shown as a quantitation standard. MwM = 1 kb Molecular weight marker. 677
Gel was loaded to optimize visualization of the degree of DNA arising from irradiation. 678
679
References 680
681
1. Nicholson WL, Munakata N, Horneck G, Melosh HJ, Setlow P. 2000. Resistance of Bacillus 682 endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev 683 64:548-72. 684
2. Klobutcher LA, Ragkousi K, Setlow P. 2006. The Bacillus subtilis spore coat provides “eat 685 resistance” during phagocytic predation by the protozoan Tetrahymena thermophila. Proceedings 686 of the National Academy of Sciences of the United States of America 103:165-170. 687
3. Driks A. 2009. The Bacillus anthracis spore. Mol Aspects Med 30:368-73. 688 4. Regis E. 1999. The biology of doom : the history of America's secret germ warfare project, 1st 689
ed. Henry Holt, New York. 690 5. Leitenberg M, Zilinskas RA. 2012. The Soviet Biological Weapons Program: A History. Harvard 691
University Press, Cambridge, MA. 692 6. Keim P, Smith KL, Keys C, Takahashi H, Kurata T, Kaufmann A. 2001. Molecular investigation 693
of the Aum Shinrikyo anthrax release in Kameido, Japan. J Clin Microbiol 39:4566-7. 694 7. Anonymous. 2010. Amerithrax Investigative Summary. Department of Justice, Government 695
USF, Washington, DC. 696 8. Rasko DA, Worsham PL, Abshire TG, Stanley ST, Bannan JD, Wilson MR, Langham RJ, Decker 697
RS, Jiang L, Read TD, Phillippy AM, Salzberg SL, Pop M, Van Ert MN, Kenefic LJ, Keim PS, 698
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
34
Fraser-Liggett CM, Ravel J. 2011. Bacillus anthracis comparative genome analysis in support of 699 the Amerithrax investigation. Proc Natl Acad Sci U S A 108:5027-32. 700
9. Driks A. 2002. Maximum shields: the assembly and function of the bacterial spore coat. Trends 701 Microbiol 10:251-4. 702
10. Bozue JA, Welkos S, Cote CK. 2015. The Bacillus anthracis Exosporium: What’s the Big 703 “Hairy” Deal? Microbiology Spectrum 3. 704
11. Stewart GC. 2015. The Exosporium Layer of Bacterial Spores: a Connection to the Environment 705 and the Infected Host. Microbiology and Molecular Biology Reviews 79:437-457. 706
12. Picard FJ, Gagnon M, Bernier MR, Parham NJ, Bastien M, Boissinot M, Peytavi R, Bergeron 707 MG. 2009. Internal control for nucleic acid testing based on the use of purified Bacillus 708 atrophaeus subsp. globigii spores. J Clin Microbiol 47:751-7. 709
13. Russell AD. 1982. The destruction of bacterial spores. Academic Press. 710 14. Horne T, Turner GC, Willis AT. 1959. Inactivation of spores of Bacillus anthracis by gamma-711
radiation. Nature 183:475-6. 712 15. Manchee RJ, Watson S. 1991. Inactivation of Vegetative Bacteria and Spores of Bacillus 713
anthracis by Gamma-Irradiation: CBDE Porton Down Technical Note No. 1090. Defence 714 Science and Technology Laboratory (Dstl), Porton Down. 715
16. Spotts Whitney EA, Beatty ME, Taylor TH, Jr., Weyant R, Sobel J, Arduino MJ, Ashford DA. 716 2003. Inactivation of Bacillus anthracis spores. Emerg Infect Dis 9:623-7. 717
17. Dang JL, Heroux K, Kearney J, Arasteh A, Gostomski M, Emanuel PA. 2001. Bacillus spore 718 inactivation methods affect detection assays. Appl Environ Microbiol 67:3665-70. 719
18. Dauphin LA, Newton BR, Rasmussen MV, Meyer RF, Bowen MD. 2008. Gamma Irradiation 720 Can Be Used To Inactivate Bacillus anthracis Spores without Compromising the Sensitivity of 721 Diagnostic Assays. Applied and Environmental Microbiology 74:4427-4433. 722
19. Melly E, Cowan AE, Setlow P. 2002. Studies on the mechanism of killing of Bacillus subtilis 723 spores by hydrogen peroxide. Journal of Applied Microbiology 93:316-325. 724
20. Young SB, Setlow P. 2003. Mechanisms of killing of Bacillus subtilis spores by hypochlorite and 725 chlorine dioxide. Journal of Applied Microbiology 95:54-67. 726
21. Aparecida da Silva Aquino K. 2012. Sterilization by Gamma Irradiation, p 171-206. In Adrovic F 727 (ed), Gamma Radiation doi:10.5772.2054. InTech, Rijeka, Croatia. 728
22. Trampuz A, Piper KE, Steckelberg JM, Patel R. 2006. Effect of gamma irradiation on viability 729 and DNA of Staphylococcus epidermidis and Escherichia coli. J Med Microbiol 55:1271-5. 730
23. Tauxe RV. 2001. Food safety and irradiation: protecting the public from foodborne infections. 731 Emerg Infect Dis 7:516-21. 732
24. Association for the Advancement of Medical Instrumentation. 2011. Sterilization of health care 733 products — Requirements and guidance for selecting a sterility assurance level (SAL) for 734 products labeled “sterile”. AAMI. Arlington, VA. 735
25. Association for the Advancement of Medical Instrumentation. 2006. Sterilization of health care 736 products—Radiation—Part 2: Establishing the sterilization dose. AAMI. Arlington, VA. 737
26. Bowen JE, Manchee RJ, Watson S, Turnbull PC. 1996. Inactivation of Bacillus anthracis 738 vegetative cells and spores by gamma irradiation. Salisbury Med Bull Suppl 87(supp):70-72. 739
27. Parisi AN, Antoine AD. 1975. Characterization of Bacillus pumilus E601 spores after single 740 sublethal gamma irradiation treatments. Appl Microbiol 29:34-9. 741
28. Neely W, Blevins W, Littrell D, Federle S. 1997. Inactivation of Biological Agent Simulants by 742 Gamma Radiation from Cobalt-60. Air Force Material Command, Eglin Air Force Base, FL. 743
29. Broomall SM, Ait Ichou M, Krepps MD, Johnsky LA, Karavis MA, Hubbard KS, Insalaco JM, 744 Betters JL, Redmond BW, Rivers BA, Liem AT, Hill JM, Fochler ET, Roth PA, Rosenzweig CN, 745 Skowronski EW, Gibbons HS. 2016. Whole-Genome Sequencing in Microbial Forensic Analysis 746 of Gamma-Irradiated Microbial Materials. Appl Environ Microbiol 82:596-607. 747
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
35
30. Majidi V. 2015. Review Committee Report: Inadvertent Shipment of Live Bacillus anthracis 748 Spores by DoD. Office of the Secretary of Defense, OASD(NCB), Defense USDo, Washington, 749 DC. 750
31. Akers J, Agalloco J. 1997. Sterility and Sterility Assurance. PDA Journal of Pharmaceutical 751 Science and Technology 51:72-77. 752
32. Herr PR. 2008. United States Postal Service: Information on the Irradiation of Federal Mail in the 753 Washington, D.C. Area. Government Accountability Office, Office GA, Washington, DC. 754
33. Niebuhr SE, Dickson JS. 2003. Destruction of Bacillus anthracis strain Sterne 34F2 spores in 755 postal envelopes by exposure to electron beam irradiation. Letters in Applied Microbiology 756 37:17-20. 757
34. Camp DW, Montgomery NK. 2008. How good labs can get wrong results - keys to accurate and 758 reproducible quantitation of Bacillus anthracis spore sampling or extraction efficiency, abstr 759 Third National Conference on Environmental Sampling and Detection for Bio-Threat Agents, Las 760 Vegas, NV, December 2008. Las Vegas, NV. 761
35. Doyle RJ, Nedjat-Haiem F, Singh JS. 1984. Hydrophobic characteristics of Bacillus spores. 762 Current Microbiology 10:329-332. 763
36. Koshikawa T, Yamazaki M, Yoshimi M, Ogawa S, Yamada A, Watabe K, Torii M. 1989. 764 Surface Hydiophobicity of Spores of Bacillus spp. Journal of General Microbiology 135:2717-765 2722. 766
37. Husmark U, Ronner U. 1990. Forces involved in adhesion of Bacillus cereus spores to solid 767 surfaces under different environmental conditions. J Appl Bacteriol 69:557-62. 768
38. Ronner U, Husmark U, Henriksson A. 1990. Adhesion of Bacillus spores in relation to 769 hydrophobicity. J Appl Bacteriol 69:550-6. 770
39. Faille C, Jullien C, Fontaine F, Bellon-Fontaine MN, Slomianny C, Benezech T. 2002. Adhesion 771 of Bacillus spores and Escherichia coli cells to inert surfaces: role of surface hydrophobicity. Can 772 J Microbiol 48:728-38. 773
40. Charlton S, Moir AJ, Baillie L, Moir A. 1999. Characterization of the exosporium of Bacillus 774 cereus. J Appl Microbiol 87:241-5. 775
41. Buhr TL, Young AA, Minter ZA, Wells CM, McPherson DC, Hooban CL, Johnson CA, Prokop 776 EJ, Crigler JR. 2012. Test method development to evaluate hot, humid air decontamination of 777 materials contaminated with Bacillus anthracis Sterne and B. thuringiensis Al Hakam spores. J 778 Appl Microbiol 113:1037-51. 779
42. Buhr TL, Young AA, Bensman M, Minter ZA, Kennihan NL, Johnson CA, Bohmke MD, 780 Borgers-Klonkowski E, Osborn EB, Avila SD, Theys AM, Jackson PJ. 2016. Hot, humid air 781 decontamination of a C-130 aircraft contaminated with spores of two acrystalliferous Bacillus 782 thuringiensis strains, surrogates for Bacillus anthracis. J Appl Microbiol 120:1074-84. 783
43. Buhr TL, Young AA, Barnette HK, Minter ZA, Kennihan NL, Johnson CA, Bohmke MD, 784 DePaola M, Cora-Lao M, Page MA. 2015. Test methods and response surface models for hot, 785 humid air decontamination of materials contaminated with dirty spores of Bacillus anthracis 786 Sterne and Bacillus thuringiensis Al Hakam. J Appl Microbiol doi:10.1111/jam.12928. 787
44. Buhr TL, Wells CM, Young AA, Minter ZA, Johnson CA, Payne AN, McPherson DC. 2013. 788 Decontamination of materials contaminated with Bacillus anthracis and Bacillus thuringiensis Al 789 Hakam spores using PES-Solid, a solid source of peracetic acid. J Appl Microbiol 115:398-408. 790
45. Titball RW, Manchee RJ. 1987. Factors affecting the germination of spores of Bacillus anthracis. 791 J Appl Bacteriol 62:269-73. 792
46. Prokop EJ, Crigler JR, Wells CM, Young AA, Buhr TL. 2014. Response surface modeling for 793 hot, humid air decontamination of materials contaminated with Bacillus anthracis Sterne and 794 Bacillus thuringiensis Al Hakam spores. AMB Express 4:21. 795
47. Buhr TL, Young AA, Minter ZA, Wells CM, Shegogue DA. 2011. Decontamination of a hard 796 surface contaminated with Bacillus anthracis ΔSterne and B. anthracis Ames spores using 797
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
36
electrochemically generated liquid-phase chlorine dioxide (eClO2). J Appl Microbiol 111:1057-798 64. 799
48. United States Centers for Disease Control and Prevention. August 14, 2017. Revised FSAP 800 Policy Statement: Inactivated Bacillus anthracis and Bacillus cereus Biovar anthracis. 801 https://www.selectagents.gov/policystatement_bacillus.html. Accessed December 12. 802
49. Wang ST, Setlow B, Conlon EM, Lyon JL, Imamura D, Sato T, Setlow P, Losick R, 803 Eichenberger P. 2006. The Forespore Line of Gene Expression in Bacillus subtilis. Journal of 804 Molecular Biology 358:16-37. 805
50. Moeller R, Raguse M, Reitz G, Okayasu R, Li Z, Klein S, Setlow P, Nicholson WL. 2014. 806 Resistance of Bacillus subtilis Spore DNA to Lethal Ionizing Radiation Damage Relies Primarily 807 on Spore Core Components and DNA Repair, with Minor Effects of Oxygen Radical 808 Detoxification. Applied and Environmental Microbiology 80:104-109. 809
51. Kohler LJ, Quirk AV, Welkos SL, Cote CK. 2017. Incorporating germination-induction into 810 decontamination strategies for bacterial spores. J Appl Microbiol doi:10.1111/jam.13600. 811
52. Driks A. 1999. Bacillus subtilis spore coat. Microbiol Mol Biol Rev 63:1-20. 812 53. Osborne JC, Miller JH, Kempner ES. 2000. Molecular mass and volume in radiation target 813
theory. Biophysical Journal 78:1698-1702. 814 54. Levinson HS, Hyatt MT. 1964. Effect of sporulation medium on heat resistance, chemical 815
composition, and germination of Bacillus megaterium spores. Journal of Bacteriology 87:876-816 886. 817
55. Moeller R, Wassmann M, Reitz G, Setlow P. 2011. Effect of radioprotective agents in sporulation 818 medium on Bacillus subtilis spore resistance to hydrogen peroxide, wet heat and germicidal and 819 environmentally relevant UV radiation. Journal of Applied Microbiology 110:1485-1494. 820
56. Buckley P, Rivers B, Katoski S, Kim MH, Kragl FJ, Broomall S, Krepps M, Skowronski EW, 821 Rosenzweig CN, Paikoff S, Emanuel P, Gibbons HS. 2012. Genetic barcodes for improved 822 environmental tracking of an anthrax simulant. Appl Environ Microbiol 78:8272-80. 823
57. Wattam AR, Abraham D, Dalay O, Disz TL, Driscoll T, Gabbard JL, Gillespie JJ, Gough R, Hix 824 D, Kenyon R, Machi D, Mao C, Nordberg EK, Olson R, Overbeek R, Pusch GD, Shukla M, 825 Schulman J, Stevens RL, Sullivan DE, Vonstein V, Warren A, Will R, Wilson MJ, Yoo HS, 826 Zhang C, Zhang Y, Sobral BW. 2014. PATRIC, the bacterial bioinformatics database and 827 analysis resource. Nucleic Acids Res 42:D581-91. 828
58. Passalacqua KD, Varadarajan A, Ondov BD, Okou DT, Zwick ME, Bergman NH. 2009. 829 Structure and complexity of a bacterial transcriptome. J Bacteriol 191:3203-11. 830
59. Passalacqua KD, Varadarajan A, Weist C, Ondov BD, Byrd B, Read TD, Bergman NH. 2012. 831 Strand-specific RNA-seq reveals ordered patterns of sense and antisense transcription in Bacillus 832 anthracis. PLoS One 7:e43350. 833
60. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR. 1989. Engineering hybrid genes without the 834 use of restriction enzymes: gene splicing by overlap extension. Gene 77:61-8. 835
61. Plaut RD, Stibitz S. 2015. Improvements to a Markerless Allelic Exchange System for Bacillus 836 anthracis. PLoS One 10:e0142758. 837
62. Buhr TL, McPherson DC, Gutting BW. 2008. Analysis of broth-cultured Bacillus atrophaeus and 838 Bacillus cereus spores. J Appl Microbiol 105:1604-13. 839
63. McCartt AD, Gates SD, Jeffries JB, Hanson RK, Joubert LM, Buhr TL. 2011. Response of 840 Bacillus thuringiensis Al Hakam Endospores to Gas Dynamic Heating in aShock Tube. 841 Zeitschrift für Physikalische Chemie 225:1367. 842
64. Stewart GS, Johnstone K, Hagelberg E, Ellar DJ. 1981. Commitment of bacterial spores to 843 germinate. A measure of the trigger reaction. Biochemical Journal 198:101-106. 844
65. Atrih A, Foster SJ. 2001. Analysis of the role of bacterial endospore cortex structure in resistance 845 properties and demonstration of its conservation amongst species. J Appl Microbiol 91:364-72. 846
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from
37
66. Kanodia S, Agarwal S, Singh P, Agarwal S, Singh P, Bhatnagar R. 2009. Biochemical 847 characterization of alanine racemase--a spore protein produced by Bacillus anthracis. BMB Rep 848 42:47-52. 849
67. Anthony KG, Strych U, Yeung KR, Shoen CS, Perez O, Krause KL, Cynamon MH, Aristoff PA, 850 Koski RA. 2011. New classes of alanine racemase inhibitors identified by high-throughput 851 screening show antimicrobial activity against Mycobacterium tuberculosis. PLoS One 6:e20374. 852
68. Bodgi L, Canet A, Pujo-Menjouet L, Lesne A, Victor JM, Foray N. 2016. Mathematical models 853 of radiation action on living cells: From the target theory to the modern approaches. A historical 854 and critical review. J Theor Biol 394:93-101. 855
856
on May 25, 2020 by guest
http://aem.asm
.org/D
ownloaded from