EPA/600/R-15/3031 December 2015 I www2.epa.gov/research
United States
Environmental Protection
Agency
Determination of the Persistence
of Non-Spore-Forming Biological
Threat Agents in the Environment
REPORT
Office of Research and Development
National Homeland Security Research Center
-------
EPA/600/R-15/303
December 2015
REPORT
Determination of the Persistence of
Non-Spore-Forming Biological Threat
Agents in the Environment
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
-------
Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's (ORD's) National Homeland Security Research Center (NHSRC), funded,
directed and managed this work through Contract Number EP-C-11-038, Task Order 0021, with
Battelle. This report has been peer and administratively reviewed and has been approved for
publication as an EPA document. The views expressed in this report are those of the authors and
do not necessarily reflect the views or policies of the Agency. Mention of trade names or
commercial products does not constitute endorsement or recommendation for use of a specific
product.
Questions concerning this document or its application should be addressed to:
M. Worth Calfee, Ph.D.
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
919-541-7600
11
-------
Contents
Disclaimer ii
Abbreviations/Acronyms v
Acknowledgments vi
Executive Summary vii
1.0 Introduction 1
2.0 Procedures 2
2.1 Biological Agents 2
2.2 Test Materials 2
2.3 Inoculation 3
2.4 Environmental Conditions and Persistence Testing Procedures 4
2.5 Recovery 5
3.0 Quality Assurance/Quality Control 7
3.1 Equipment Calibration 7
3.2 QC Results 7
3.3 Audits 9
3.4 QA/QC Reporting 9
3.5 Data Review 9
4.0 Persistence Test Results 10
4.1 Method Demonstration - Organism Recovery 10
4.2 Persistence Results 10
5.0 Summary 22
6.0 References 24
Figures
Figure 4-1. Y. pestis Persistence at 5 ± 2 °C and 30 ± 15% RH 12
Figure 4-2. Y. pestis Persistence at 22 ± 2 °C and 40 ± 15% RH 13
Figure 4-3. Y. pestis Persistence at 35 ± 2 °C and 65 ± 15% RH 13
Figure 4-4. F. tularensis Persistence at 5 ± 2 °C and 30 ± 15% RH 16
Figure 4-5. F. tularensis Persistence at 22 ± 2 °C and 40 ± 15% RH 16
Figure 4-6. F. tularensis Persistence at 35 ± 2 °C and 65 ± 15% RH 17
Figure 4-7. B. mallei Persistence at 5 ± 2 °C and 30 ± 15% RH 19
Figure 4-8. B. mallei Persistence at 22 ± 2 °C and 40 ± 15% RH 20
Figure 4-9. B. mallei Persistence at 35 ± 2 °C and 65 ± 15% RH 20
Tables
Table ES-1. Summary of Persistence Testing on Glass (Non-Lyophilized) viii
Table ES-2. Summary of Persistence Testing on Glass (Lyophilized) viii
Table ES-3. Summary of Persistence Testing on Soil (Non-Lyophilized) ix
Table ES-4. Summary of Persistence Testing on Soil (Lyophilized) ix
Table 2-1. Test Materials 3
Table 2-2. Soil Sample Analysis 3
Table 2-3. Media Types and Incubation Parameters 4
Table 3-1. Positive Control Percent Recoveries 7
Table 3-2. Persistence Testing Operational Parameters 8
Table 3-3. Performance Evaluation Audits 9
Table 4-1. Recovery Data from Method Demonstration 10
Table 4-2. Y. pestis Persistence on Glass (Non-Lyophilized) 11
iii
-------
Table 4-3. Y. pestis Persistence on Glass using a Lyophilized Inoculum 12
Table 4-4. Qualitative Y. pestis Persistence in Soil 14
Table 4-5. F. tularemis Persistence on Glass Using a Non-Lyophilized Inoculum 15
Table 4-6. F. tularensis Persistence on Glass Using a Lyophilized Inoculum 15
Table 4-7. Qualitative F. tularensis Persistence in Soil 17
Table 4-8. B. mallei Persistence on Glass Using a Non-Lyophilized Inoculum 18
Table 4-9. B. mallei Persistence on Glass Using a Lyophilized Inoculum 19
Table 4-10. Qualitative B. mallei Persistence in Soil 21
Table 5-1. Minimum Exposure Times for Organism Recovery on Glass or Soil 22
IV
-------
Abbreviations/Acronyms
B. mallei Burkholderia mallei
BSC biological safety cabinet
°C degrees Celsius
CPU colony forming unit(s)
CI confidence interval
CMAD Consequence Management Advisory Division
EPA U.S. Environmental Protection Agency
F. tularensis Francisella tularensis
g gravity
g gram(s)
HSRP Homeland Security Research Program
kg Kilogram(s)
kGy kilogray(s)
mg milligram(s)
mL milliliter(s)
mm millimeter(s)
(iL microliter(s)
MFDB microbial freeze dry buffer
NHSRC National Homeland Security Research Center
NIST National Institute of Standards and Technology
OEM Office of Emergency Management
ORD Office of Research and Development
PBS phosphate-buffered saline
PCR polymerase chain reaction
QA quality assurance
QAPP Quality Assurance Project Plan
QC quality control
QMP quality management plan
RH relative humidity
rpm revolution(s) per minute
SD standard deviation
SE standard error
TSA tryptic soy agar
TSB tryptic soy broth
Y. pestis Yersinia pestis
-------
Acknowledgments
Contributions of the following individuals and organizations to this report are gratefully
acknowledged:
Project Team
Dr. M. Worth Calfee (Principal Investigator; ORD, NHSRC)
Elise Jakabhazy (OEM, CMAD)
John Martin (Region 6)
Leroy Mickelsen (OEM, CMAD)
Mike Nalipinski (OEM, CMAD)
Erin Silvestri (ORD, NHSRC)
Peer reviewers
Dr. Gene Rice (US EPA, NHSRC), Dr. Morgan Minyard (Department of Defense, Defense
Threat Reduction Agency), Dr. Tim Dean (US EPA, Air Pollution Prevention Control Division)
Battelle Memorial Institute
VI
-------
Executive Summary
The persistence of biological agents outside a host is influenced by environmental conditions and
the materials with which these biological agents are in contact. This generation of scientifically
defensible persistence data is useful for the proper planning of decontamination efficacy tests and
for formulation of response or remediation plans in preparation for possible natural occurrences or
intentional releases of biological agents. This report presents the results of an investigation to
evaluate the persistence (or natural attenuation) of Yersiniapestis (Y. pestis), Francisella tularensis
(F. tularensis), and Burkholderia mallei (B. mallei) on glass and soil under multiple environmental
conditions and time points.
Persistence (recovery of viable organisms) was assessed for Y. pestis, F. tularensis, and B. mallei
inoculated onto glass coupons and soil materials. The cell suspensions deposited onto the surfaces
were allowed to dry under ambient conditions for approximately one hour or were lyophilized for
approximately two hours, prior to being placed into environmental chambers for up to 99 days.
The target environmental conditions were low temperature (5 degrees Celsius [°C]) and low
relative humidity (RH; 30%), moderate temperature (22 °C) and moderate RH (40%), and high
temperature (35 °C) and high RH (65%) conditions.
Summary of Results
When inoculated coupons were lyophilized and maintained at a low temperature and RH, all three
organisms survived on glass at a higher concentration than when inoculated and dried under
ambient conditions (non-lyophilized). A similar effect was observed at the moderate temperature
conditions, but with fewer organisms recovered at each time point. F. tularensis survived >63 days
(<99 days), and B. mallei survived >6 day (<21 days) when using a non-lyophilized inoculum,
while viable cells were recovered for all three organisms after 99 days when lyophilized. Less than
1 log (logic) reduction was observed for all three organisms on glass when lyophilized and held at
the low temperature and RH condition. At the high temperature condition, F. tularensis survived
>6 days (<21 days) and B. mallei > 1 day (<6 days) using a non-lyophilized inoculum, while no
viable Y. pestis was recovered at any time point. When lyophilized, all three organisms survived
>1 day (<6 days) at the high temperature/RH condition. Tables ES-1 and ES-2 summarize the
quantitative recoveries of Y. pestis, F. tularensis, and B. mallei applied to 5 x 5 millimeter (mm)
glass coupons. Similar results were observed with 100 milligram (mg) soil samples, but these
samples were only qualitatively assessed due to indigenous flora present in the soil samples that
interfered with organism enumeration (Tables ES-3 and ES-4).
vn
-------
Table ES-1. Summary of Persistence Testing on Glass (Non-Lyophilized)
Test Temperature %
Organism "C1' KIT
_ . Mean Recovered Organisms (log survivorship) by Duration in
Starting
Inoculum*
5 ± 3 °C; 30 ± 15% RH
Y. pestis
F. tularensis
B. mallei
Y. pestis
F. tularensis
B. mallei
Y. pestis
F. tularensis
B. mallei
6
6
6
23
22
23
35
35
35
41
34
38
46
48
47
70
67
65
7.50
8.17
8.06
7.50
8.17
8.06
7.50
8.17
8.06
5.80
6.93
8.06
22 ± 3 °C; 40
5.80
6.93
8.06
35 ± 3 °C; 65
5.80
6.93
8.06
4.88
7.93
5.82
± 15% RH
3.88
7.24
5.92
± 15% RH
ND
1.16
3.19
4.55
7.16
5.93
1.26
4.74
0.36
ND
0.16
ND
4.15
6.75
4.77
ND
2.34
ND
ND
ND
3.71
5.10
1.74
ND
0.16
ND
ND
3.46
4.62
0.10
ND
ND
ND
ND
*Data are expressed as mean logs colony forming units (CPU) recovered.
"—" indicates no data collected at indicated time point.
tMean temperature and RH values based on continuous monitoring at six minute intervals over the entire 99 days.
"ND" indicates that no viable organisms were recovered from any of the replicate coupons.
Table ES-2. Summary of Persistence Testing on Glass (Lyophilized)
Test Temperature %
Organism "C RHf
Mean Recovered Organisms (log survivorship) by Duration in
Starting^ Days*
5 ± 3 °C; 30 ± 15% RH
Y. pestis
F. tularensis
B. mallei
Y. pestis
F. tularensis
B. mallei
Y. pestis
F. tularensis
B. mallei
6
6
6
23
22
23
35
35
35
41
34
38
46
48
47
70
67
65
8.35
8.46
7.98
8.35
8.46
7.98
8.35
8.46
7.98
8.59
7.40
7.70
22 ± 3 °C; 40
8.59
7.40
7.70
35 ± 3 °C; 65
8.59
7.40
7.70
8.66
7.63
7.41
± 15% RH
8.48
7.27
6.63
± 15% RH
1.82
4.99
5.64
8.13
7.48
7.51
8.01
6.95
5.91
ND
ND
ND
8.31
7.52
7.27
7.01
6.51
6.41
ND
8.51
7.29
7.12
4.92
6.82
6.01
ND
7.97
7.63
7.16
2.69
6.02
5.53
ND
*Data are expressed as mean logs CFU recovered.
"—" indicates no data collected at indicated time point.
tMean temperature and RH values based on continuous monitoring at six minute intervals over the entire 99 days.
"ND" indicates that no viable organisms were recovered from any of the replicate coupons.
Vlll
-------
Table ES-3. Summary of Persistence Testing on Soil (Non-Lyophilized)
Test Temperature %
Organism "C1' RH^
Starting Recovered Organisms by Duration in Days
5 ± 3 °C; 30 ± 15% RH
Y. pestis
F. tularensis
B. mallei
6
6
6
41
34
38
7.50 •/ S S ~
8.17 ^ ^ ^ ^
8.06 ^ ^ ^ ^
; ;
22 ±3 °C; 40 ± 15% RH
Y. pestis
F. tularensis
B. mallei
23
22
23
46
48
47
7.50
-------
1.0 Introduction
The U.S. Environmental Protection Agency's (EPA's) Homeland Security Research Program
(HSRP) is helping to protect human health and the environment from adverse impacts resulting
from the release of chemical, biological, or radiological agents. With an emphasis on
decontamination and consequence management, water infrastructure protection, and threat and
consequence assessment, the HSRP is working to develop tools and information that will help
detect the intentional introduction of chemical or biological contaminants into buildings or water
systems; contain these contaminants; decontaminate buildings, outdoor environments, or water
systems; and facilitate the disposal of material resulting from restoration activities.
In this work, the persistence of three biological threat agents (Yersinia pestis [Y. pestis],
Francisella tularensis [F. tularensis], and Burkholderia mallei [B. mallei]) was evaluated under
varying environmental conditions to assist in determining conditions under which further
decontamination may likely be needed. The results of this investigation provide technology users
and stakeholders with high quality, peer-reviewed data on the effectiveness of various temperature
and relative humidity (RH) combinations on indoor and outdoor materials (glass and soil)
contaminated with these biological agents. Soil and glass were chosen to represent two common
yet vastly different surface types common within the environment.
-------
2.0 Procedures
Glass (5x5 millimeter [mm]) and soil (100 milligrams [mg]) were inoculated with either 7 pestis,
F. tularensis, or B. mallei (-3.17 x 109 to2.87 x 1010 colony forming units/milliliter [CFU/mL]),
allowed to air dry or were lyophilized on the material, and then exposed to three environmental
conditions (5 ± 3 °C and 30 ± 15% RH, 22 ± 3 °C and 40 ± 15% RH, or 35 ± 3 °C and 65 ± 15%
RH) for controlled exposure durations up to 99 days. Persistence was determined quantitatively
for glass and qualitatively for soil (due to the abundance of indigenous flora observed in the soil
samples, making it difficult to enumerate) for each environmental condition at 0, 1, 6, 21, 63, and
99 days. Persistence was assessed by measuring the recovery of Y. pestis, F. tularensis, and B.
mallei as CPU from each tested combination of environmental condition and exposure duration.
All testing was performed in accordance with the peer-reviewed and EPA-approved Quality
Assurance Project Plan (QAPP).
2.1 Biological Agents
Testing was conducted with virulent Y. pestis CO92 (BEI Resources, Manassas, VA, NR-641), F.
tularensis SCHU S4 (BEI Resources, NR-10492), and B. mallei China 7 (BEI Resources, NR-23).
All three are Gram-negative, non-spore-forming coccobacilli. All samples were maintained in pure
culture for this investigation. Ribonucleic acid from stock cultures was isolated using a NucliSens®
easyMAG™ (bioMerieux, St. Louis, MO) and were identified by polymerase chain reaction (PCR)
using a 7900HT Fast Real-Time PCR System (Applied Biosystems, Grand Island, NY) using
internal standard operating procedures with specialized primers and probes. All cultures were
confirmed to be either 7 pestis CO92, F. tularensis SCHU S4, or B. mallei China 7 by PCR as
well as Gram stain.
2.2 Test Materials
Information on the materials and sterilization approaches used for testing are presented in Table
2-1. Persistence testing was conducted on two materials: glass and soil. These materials were
selected to represent common nonporous (glass) and porous (soil) materials or matrices. Glass
materials were supplied by the manufacturer at a uniform length and width (5^5 mm) from stock
material. Soil was supplied by a commercial vendor and approximately 100 mg aliquotted as
needed into 1.5 mL microcentrifuge tubes. The selected sterilization approach, shown in Table 2-
1, was based on cost-effectiveness and minimization of the physical alterations of the materials.
Autoclaving was conducted at 121 °C for 15 minutes and electron-beam sterilization was
conducted at -200 kilograys (kGy) by E-Beam Services, Inc. (Lebanon, OH) using a 5.0 MeV E-
beam accelerator. The intent of sterilization was to eliminate the probability of bacterial
background interference. Approximately 6 kilograms (kg) of soil were sent to E-Beam Services,
Inc., for irradiation in -500 gram (g) aliquots. Following receipt of soil samples after irradiation,
a representative set of samples (two 100 mg samples from each 500 g lot) was extracted, plated
onto tryptic soy agar (TSA) and confirmed to be sterile. However, for the t=0 time point during
the 7 pestis testing, contamination (indigenous flora, not the test organisms) was noted in soil
samples during enumerations. As result, subsequent soil samples were assessed only qualitatively.
-------
Table 2-1. Test Materials
Material
Glass
Soil
Lot or Batch No.
EPA-1101
Mollisol, HCB-SL-PF,
collected from Easter, ND
Supplier
Erie Scientific
(Portsmouth, NH)
Agvise Laboratories
(Northwood, ND)
Approximate Size
5x5x1 mm
lOOmginl.SmL
microcentrifuge tubes
Material
Preparation
Autoclave
E-Beam
Soil samples (500 g, pre- and post-sterilization via E-beam at -200 kGy) were sent to an
independent laboratory for analysis of moisture, organic content and other characteristics. An
approximately 500 g sample was analyzed in compliance with 40 CFR Part 160 for Good
Laboratory Practices for the parameters listed in Table 2-2.
Table 2-2. Soil Sample Analysis
33
Cation Exchange Capacit
4/100 g
22.0
24.3
29
loisture at 1/3 Br
:ld Capacit)
42.0
36.9
35
Bulk Density (g/c
Clay Loam Clay Loam
7.22
7.21
0.81
7.6
=
7.4
71.7 3150 70.7 3440 15.3 402
15.6 456 0.3 13 0.3 19
=
4.4
374
4.4
418
8.4
18
8.9
'Pre = Pre-Sterilization
2E = E-beam irradiation @ 200 kGy
3cc = cubic centimeters
4meq = milliequivalents
2.3 Inoculation
Fresh cultures were prepared in advance of each day that coupons were inoculated by transferring
colonies from a streak plate (freshly growing or stored less than 2 weeks at 2 °C to 8 °C) into 100
mL of the appropriate liquid culture media (Table 2-3). This culture was then incubated overnight
at the appropriate temperature (Table 2-3) on an orbital shaker set to 200 revolutions per minute
(rpm). The bacterial culture was then centrifuged at 10,000 x gravity (g) for 10 minutes, the
supernatant discarded, and the pellet resuspended into 10 mL of either the appropriate buffer
(Table 2-3) for the non-lyophilized inoculum or into microbial freeze dry buffer (MFDB; OPS
Diagnostics, Lebanon, NJ). All samples were inoculated with one 10 microliter (jiL) drop of
concentrated agent. The non-lyophilized samples were allowed to dry under ambient conditions
for one hour prior to extraction (Time 0 control) or placed into the respective environmental
-------
chambers. The lyophilized samples were placed at <-70 °C overnight prior to being placed in the
lyophilizer (Labconco 77530, Kansas City, MO) for approximately two hours. These samples were
then extracted (Time 0 control) or placed into their respective environmental chambers. All
inoculated and dried glass coupons were kept in 1.5 mL microcentrifuge tubes for ease of handling.
Table 2-3. Media Types and Incubation Parameters
Organism (strain)
Solid Media
Liquid Media
Y. pestis (CO92)
F. tularensis (SCHU S4)
B. mallei (China 7)
Tryptic Soy Agar
(G60, Hardy Diagnostics,
Santa Maria, CA)
Chocolate II
(221169, BD Biosciences,
Franklin Lakes, NJ)
Chocolate II
Tryptic Soy Broth
(T1550, Teknova,
Hollister, CA)
Mueller Hinton Broth
(90922, Sigma Aldrich,
St. Louis, MO)
Nutrient Broth
(233000, BD Biosciences)
Incubation
Temperature
26 ±2
37 ±2
37 ±2
Incubation
Time (hours)
36-96
36-96
48-96
2.4 Environmental Conditions and Persistence Testing Procedures
Persistence testing was conducted under three sets of target environmental conditions:
• Low temperature and relative humidity: 5 ± 3 °C and 30 ± 15% RH
• Moderate temperature and relative humidity: 22 ± 3 °C and 40 ± 15% RH
• High temperature and relative humidity: 35 ± 3 °C and 65 ± 15% RH.
All actual values for environmental conditions are presented in Table 3-2. The test coupons were
held at the required temperature for the required time period (up to 99 days) in a closed, airtight
persistence testing chamber (Lock & Lock, HPL838P, Farmers Branch, TX). All lids to the 1.5
mL microcentrifuge tubes were left open for the duration of the required time period. After the
coupons were removed from their respective persistence testing chambers, all were moved to a
biological safety cabinet (BSC) II for extraction (refer to Section 2.5).
For all testing conditions, fixed humidity-point salt solutions (magnesium chloride for the low
temperature condition, potassium carbonate for the moderate temperature condition, and
potassium iodide for the high temperature condition) were used to maintain the RH in the
respective chambers. All salts were mixed with water and added as a slurry to separate containers
and placed in the bottom of the appropriate persistence test chambers following ASTM E104.(1)
The coupons were never allowed to come into direct contact with the salt or the salt containers.
For the low temperature and RH (5 ± 3 °C and 30 ± 15% RH) test, persistence coupons were kept
in a refrigerator; chambers containing the moderate and high temperature and RH (22 ± 3 °C and
40 ± 15% RH, and 35 ± 3 °C and 65 ± 15% RH) were kept in an incubator set to the appropriate
temperature. The temperature and RH for all conditions were recorded approximately every six
minutes using a HOBO data logger (Onset Computer Corporation, Bourne, MA). The actual
temperature and RH levels observed during testing are documented in Section 3.
-------
2.5 Recovery
For sample extraction, glass materials were placed into 1.5 mL microcentrifuge tubes containing
1 mL sterile phosphate buffered saline (PBS), and 1 mL of sterile PBS was added to each 100 mg
soil sample. All vials were vortexed for approximately two minutes at room temperature and then
sonicated (Bransonic 1200, Danbury, CT) for approximately two minutes at room temperature.
Following extraction, a series of dilutions (serial 1:10 dilutions) was prepared using PBS. Aliquots
(0.1 mL) of the undiluted extract and dilutions were plated as appropriate in triplicate onto the
specified solid agar for each organism. The cultures were incubated for the appropriate time and
temperature according to Table 2-3. The colonies were counted manually, and the concentration
of viable organisms determined in CFU/mL. Typically, plates having colony counts between 25
and 250 are used for calculating the CFU/mL. Under certain circumstances (i.e., poor recovery,
reduced persistence over time), there were fewer than 25 colonies per plate from the undiluted
extract. In these cases, the number of colonies was counted and recorded even if there were fewer
than 25 colonies per plate. The total CFU/coupon was calculated as:
Total CFU/coupon = [(mean CPU plate count x L'dilution factor)Vplatedvolume] (1)
where:
Mean CFU plate count = average number of colonies counted on three
replicate plates
Plated volume =0.1 mL
Dilution factor = portion of the total extraction buffer that was used
to prepare the dilutions
A single viable bacterium present in a plated aliquot of sample under ideal growth conditions
would be expected to be observed as one CFU. Therefore, the individual coupon detection limit is
approximately 3.3 CFU/coupon if one CFU was observed on one of three plates of the undiluted
extract. Since only a portion (i.e., 0.1 mL aliquot per plate) of undiluted extract is cultured, viable
bacteria could be present in the extract that were not used for plating (approximately 0.9 mL).
However, given the number of replicate coupons (five) and replicate plates (three) per undiluted
coupon extract, there is a low probability the presence of viable bacteria would go undetected.
The recovery of bacteria (quantified as mean CFU/coupon ± standard deviation [SD]) was
calculated for each environmental condition and exposure duration combination by dividing the
total number of viable organisms extracted from all five test coupons by the number of replicate
coupons (i.e., five). Attenuation refers to the log reduction (as calculated by Equation 2) of
persistence test coupons held at their respective environmental conditions for up to 99 days.
Reduction for a test organism on the /'th coupon material was calculated as the difference between
those mean log values:
Attenuation = (log CFUci}) - (log CFUttJ) (2)
where log CFUcy refers to they individual logarithm values obtained from the positive control
coupons and log CFUty refers to they individual logarithm values obtained from the corresponding
test coupons, and the overbar designates a mean value. In tests conducted under this plan, there
were five control and five corresponding test coupons (i.e.,7 = 5). In the case where no CFU were
-------
found in a coupon extract, a CPU value of 1 was assigned, resulting in a log CPU of zero for that
coupon.
The variances (i.e., the square of the SD) of the log CFUcy and log CFUty values were also
calculated for both the control and test coupons (i.e., S2cy and *$%•), and were used to calculate the
pooled standard error (SE) for the efficacy value calculated in Equation 3, as follows:
(3)
where the number 5 again represents the number y of coupons in both the control and test data sets.
Thus, each result is reported as a log reduction value with an associated SE value.
The significance of differences in attenuation across the different environmental conditions and
inoculation procedures was assessed based on the 95% confidence interval (CI) as:
95% CI = Efficacy ± (1.96 x SE) (4)
For extraction of soil samples, 1 mL of PBS was added to each microcentrifuge tube and extracted
as stated above. However, these samples were plated on selective media (undiluted sample only)
to assess the presence of the target organism. Y. pestis samples were plated on CIN agar (BD
Biosciences; Cat # 221848), F. tularemis samples were plated on Modified Thayer Martin agar
(BD Biosciences; Cat #221567), and B. mallei samples were plated on Burkholderia Cepacia agar
(Remel Cat #R01709). Target organisms were visually confirmed, and no suspect organisms were
noted.
-------
3.0 Quality Assurance/Quality Control
Quality assurance (QA)/quality control (QC) procedures were performed in accordance with the
Battelle Quality Management Plan (QMP) and the QAPP/1^ The QA/QC procedures and results
are summarized below.
3.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, biological safety cabinets) and monitoring devices (e.g.,
thermometer, hygrometer) used at the time of evaluation were verified as being certified,
calibrated, or validated.
3.2 QC Results
QC efforts conducted during testing included test material method demonstration (i.e., extraction
efficiency), positive control samples (inoculated and extracted at Time 0) and procedural blanks
(not inoculated and extracted at each time point).
Results for all positive control samples were within the target recovery range of 0.1% to 120% of
the inoculated organisms, except where indicated in Table 3-1. Unfortunately, with the nature of
vegetative bacteria, the counts are not available for up to 96 hours post-inoculation, precluding a
restart of the test.
Table 3-1. Positive Control Percent Recoveries (Glass only)
Organism
Y. pestis
F. tularensis
B. mallei
Inoculation Method
Non-lyophilized
Lyophilized
Non-lyophilized
Lyophilized
Non-lyophilized
Lyophilized
Mean Logs Observed
5.80
8.59
6.93
7.40
8.06
7.70
Mean % Recovery
2.02
189.42*
9.58
10.29
98.97
51.88
*Positive control sample outside the allowable 0.1 to 120% of inoculated levels.
All procedural blanks met the criterion of no observed CPU for all organisms tested.
3.2.1 Operational Parameters
The temperature and RH during each test were controlled and recorded, as described in Section
2.4. Readings were taken once every six minutes for the duration of the contact time. The actual
operational parameters for each test are shown in Table 3-2 and reported as the average value ±
SD. A rise and/or fall of temperature and RH was observed when extracting coupons from the
persistence testing chambers at the respective time points. However, all coupons returned to the
appropriate target testing condition within one hour of opening the chambers, and any change in
temperature or RH is expected to have no impact on the results as the time-weighted average
conditions were well within the target ranges. In some instances, samples were not collected at
later time points as earlier time points showed complete inactivation of the organism.
-------
Table 3-2. Persistence Testing Operational Parameters
Contact Target Environmental Conditions
Organism
Y. pestis
F. tularensis
B. mallei
(Da^s) Temperature (°C)
1
6
21 5±3
63
99
1
6
21 22 ±3
63
99
1
6
21 35 ±3
63
99
1
6
21 5±3
63
99
1
6
21 22 ±3
63
99
1
6
21 35 ±3
63
99
1
6
21 5±3
63
99
1
6
21 22 ±3
63
99
1
6
21 35 ±3
63
99
RH (%)
30 ±15
40 ±15
65 ±15
30 ±15
40 ±15
65 ±15
30 ±15
40 ±15
65 ±15
Actual Environmental Conditions
Temperature (°C)
6.18 ±2.38
5.46 ±1.07
5.64 ±0.81
5.65 ±0.52
5.75 ±0.54
21.88 ±0.12
22.38 ±0.67
22.59 ±0.38
22.51 ±0.57
22.52 ±0.50
34.57 ±2.66
35.26 ±1.19
32.25 ±1.23
35.41 ±0.42
35.42 ±0.40
6.30 ±2.63
6.43 ± 1.21
6.23 ± 0.76
5.91 ±0.75
6.09 ±0.67
22.71 ±0.07
22.63 ± 0.07
22.38 ±0.45
22.47 ± 0.27
22.47 ± 0.22
35. 14 ±0.69
35.26 ±0.72
35.36 ±0.55
—
—
6.54 ±3. 15
6.82 ±1.55
5.92 ±1.14
6.49 ±0.83
6.25 ±0.7
22.46 ± 0.47
22.59 ±0.20
22.71 ±0.14
22.69 ±0.29
22.67 ± 0.24
34.65 ±3.03
35.28 ± 1.42
—
—
-
RH (%)
43. 11 ±1.78
39.32 ±3.28
42.76 ± 2.86
39.67 ±3. 19
40.61 ±3.28
54.31 ±2.01
49.98 ±2.30
49.32 ±1.31
45.88 ±1.72
45.76 ±1.53
70.67 ± 5.67
69.43 ±2.32
69.38 ±2.54
69.74 ±0.68
69.77 ± 0.66
39.89 ±5.58
34.64 ±3. 39
34.15 ±2.37
33.73 ±1.59
33.77 ± 1.37
47.48 ±0.82
47.33 ±0.65
47.56 ±0.78
47.74 ±0.53
47.93 ±0.61
65.84 ±3. 31
66.92 ±2. 18
66.92 ± 1.42
—
—
44.64 ±3. 86
39.99 ±2.62
37.47 ±2.44
38.96 ±1.80
37.75 ±1.18
47.80 ±0.99
46.78 ± 0.64
46.99 ±0.40
47.01 ±0.39
47.05 ±0.34
64.58 ±5.13
64.64 ± 2.29
—
—
-
~ denotes that samples were not collected; therefore, no temperature and RH data are available.
-------
3.3 Audits
3.3.1 Performance Evaluation Audit
Performance evaluation audits were conducted to assess the quality of the results obtained during
these tests. Table 3-3 summarizes the performance evaluation audits that were performed.
Table 3-3. Performance Evaluation Audits
Measurement
Volume
Audit Procedure
Micropipettes checked by gravimetric
evaluation
Allowable
Tolerance
±10%
Actual Tolerance
14 pipettes checked, all <8%
Compared to independent National
Temperature Institute of Standards and Technology
(NIST)-traceable thermometer value
±2°C
187 temperature comparisons,
all differences were <0.2 °C
RH
Compared to independent NIST-
traceable hygrometer value
±10%
187 RH comparisons, all
differences were <2%
Time
Compared to independent NIST-
traceable timer value
2 seconds/hour 0 seconds/hour for 5 instances
3.3.2 Technical Systems Audit
Observations and findings from the technical systems audit were documented and submitted to the
laboratory staff lead for response. Audits were conducted by the Battelle Quality Assurance
Officer on January 14, 2015, to ensure the tests were being conducted in accordance with the
QAPP and Battelle QMP. As part of the audit, test procedures were compared to those specified in
the QAPP, and data acquisition and handling procedures were reviewed. None of the findings of
the technical systems audit required corrective action.
3.3.3 Data Quality Audit
All transcribed data (100%) and 10% of all calculations and electronically transferred data
acquired during the evaluation were audited. A QA auditor traced the data from the initial
acquisition, through reduction and statistical analysis, to final reporting to ensure the integrity of
the reported results. All calculations performed on the data undergoing the audit were checked.
3.4 QA/QC Reporting
Each assessment and audit was documented in accordance with the QAPP and Battelle QMP. For
these tests, findings were noted (none significant) in the data quality audit, but no follow-up
corrective action was necessary. The findings were mostly minor data transcription errors requiring
some recalculation of efficacy results, but none were gross errors in recording. Copies of the
assessment reports were distributed to the Battelle Contract Manager and laboratory staff. QA/QC
procedures were performed in accordance with the QAPP.
3.5 Data Review
Records and data generated in the evaluation received a QC/technical review before they were
utilized in calculating or evaluating results and prior to incorporation in reports. This process
consists of review of all data transcribed from laboratory worksheets into Excel spreadsheets as
well as embedded calculations for reportable values. The process also consists of reviewing
general laboratory documentation according to Battelle standard operating procedures.
-------
4.0 Persistence Test Results
For this investigation, persistence data were generated for Y. pestis, F. tularensis, and B. mallei in
contact with glass and soil materials exposed to three environmental conditions for controlled
exposure durations up to 99 days. Persistence curves were generated, where applicable, by
graphing the log survivorship of each material against time in days for each set of environmental
condition and organism combination. The following sections summarize the results of the method
demonstration (tests conducted initially to ensure sufficient organism recovery) and the persistence
investigation.
4.1 Method Demonstration - Organism Recovery
A method demonstration was performed with each organism to confirm the methods used for
extraction were sufficient for this application. Additionally, three buffers [tryptic soy broth (TSB),
microbial freeze dry buffer (MFDB), and 6% lactose] were tested to ensure the buffer chosen
yielded sufficient recovery for each organism following the lyophilization process. TSB was
chosen to compare as it is the buffer used in wet inoculum, and the latter two chosen as routinely
used lyophilization buffers/2'3) Briefly, samples were prepared as described in Section 2.3, except
the inoculation material was prepared in three preparations: TSB, MFDB, and 6% lactose.
Materials were inoculated and allowed to dry for one hour (control) or frozen and lyophilized in
each of the three preparations (n = 5). Samples were extracted as described in Section 2.5, and the
results are summarized in Table 4-1. MFDB was chosen for all three organisms for the preparation
of the samples for lyophilization based on these data and for consistency among the three
organisms.
Table 4-1. Recovery Data from Method Demonstration (Glass only)
Organism
Y. pestis
F. tularensis
B. mallei
Test Method/Buffer
Non-Lyophilized/TSBi
Lyophilized/TSB
Lyophilized/MFDBt
Lyophilized/6% Lactose
Non-Lyophilized/TSB
Lyophilized/TSB
Lyophilized/MFDB
Lyophilized/6% Lactose
Non-Lyophilized/TSB
Lyophilized/TSB
Lyophilized/MFDB
Lyophilized/6% Lactose
noculation Control
CFU/coupon
5.13 >
1.34 >
2.11 >
2.29 >
3.13 >
3.73 >
3.07 >
1.60 >
1.35 >
1.40 >
1.37 >
9.40 >
< 106
< 107
< 107
< 107
< 107
<108
<108
<108
<108
<109
<109
<108
CFU/coupon*
1.89 ±0.81 >
2.45 ±1.41 >
4.83 ±4.89 >
3.75 ±3.85 >
4.20 ±3.39 >
9.77 ±3.83 >
1.08 ±0.36 >
4.43 ± 1.42 >
8.61 ±2.28 >
1.15 ±0.24 >
7.23 ±1.16 >
7.18 ±4.05 >
•
< 106
< 106
<108
< 107
< 104
< 106
< 106
< 106
%*
3.69 ±1.57
0.018 ±0.011
228.70 ±23 1.86
16.39 ±16.81
13.42 ±10.82
2.62 ±1.03
35.33 ±11. 86
27.71 ± 8.87
0.064 ±0.017
0.082 ±0.017
0.53 ±0.08
0.76 ±0.43
*Data are expressed as mean ± standard deviation of five replicate coupons.
*TSB = tryptic soy broth
t MFDB = microbial freeze dry buffer
4.2 Persistence Results
Persistence results for each organism/environmental condition combination are summarized in
Tables 4-2 through 4-10 and Figures 4-1 through 4-9.
10
-------
4.2.2 Y.pestis
The results obtained for 7 pestis persistence on glass are summarized in Tables 4-2 and 4-3 and
Figures 4-1 through 4-3. The qualitative persistence in soil is summarized in Table 4-4. When
lyophilized (Table 4-3), 7 pestis persisted on glass for at least 99 days under both low and
moderate temperature and RH conditions (>7 logs and >2 logio recovery, respectively). For non-
lyophilized samples (Table 4-2), 7 pestis persisted for at least 99 days at the low temperature/RH
conditions, but with lower recoveries (3.46 logs viable organisms recovered). At the moderate
temperature/RH condition, the non-lyophilized 7 pestis was recoverable after six days.
Additionally, no 7 pestis was recovered from glass for any time point when not lyophilized and
only at day one when lyophilized 7 pestis was tested at the high temperature/RH condition.
Similar to the results observed from glass, 7 pestis persisted up to 99 days under the low
environmental conditions and >6 days under the moderate environmental conditions with both
preparations in soil. No organisms were detected after six days in soil under the high temperature
environmental conditions. Analysis of Day 21 samples was not completed, as contamination of
soil samples with indigenous bacteria was detected, and revised methods (qualitative results only,
exploration of selective media) were being developed. As a result, data from this time point were
not collected.
Table 4-2. Y. pestis Persistence on Glass (Non-Lyophilized)
„ ,. Inoculation
Duration „ , ,
Control
(Days) (CFU/coupon)
Mean Recovered Y. pestis, Log Survivorship*
Positive Control Test Coupon*
Mean Log
Reduction8
5 ± 3 °C; 30 ± 15% RH
1
6
21 3.17xl07
63
99
1
6
21 3.17xl07
63
99
4.88 ±0.15
4.55 ±0.32
5. 80 ±0.06 4.15 ±0.19
3.71 ±2.01
3.46 ±0.43
22 ± 3 °C; 40 ± 15% RH
3. 88 ±0.17
1.26 ±0.82
5. 80 ±0.06 0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.92 ±0.14
1.26 ±0.28
1.65 ±0.17
2.09 ±1.76
2.34 ±0.38
1.92 ±0.16
4.54 ±0.72
5. 80 ±0.05
5. 80 ±0.05
5. 80 ±0.05
35 ± 3 °C; 65 ± 15% RH
1
6
21 3.17xl07
63
99
0.00 ±0.00
0.00 ±0.00
5. 80 ±0.06 0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
5. 80 ±0.05
5. 80 ±0.05
5. 80 ±0.05
5. 80 ±0.05
5. 80 ±0.05
*Data are expressed as mean log survivorship ± standard deviation.
tPositive control coupons were inoculated and extracted at time zero (i.e., one hour after inoculation); one set of positive controls
was used for all test durations.
*Test coupons were inoculated and exposed to the environmental condition for the exposure duration.
§Data are expressed as mean log reduction ± 95% confidence interval (CI).
11
-------
Table 4-3. F. pestis Persistence on Glass using a Lyophilized Inoculum
„ ,. Inoculation
Duration „ , ,
~ , Control
( yS) (CFU/coupon)
Mean Recovered Y. pestis, Log Survivorship*
Positive Control Test Coupon*
Mean Log
Reduction8
5 ± 3 °C; 30 ± 15% RH
1
6
21 2.25 x 108
63
99
8.66 ±0.11
8.13 ±0.48
8.59 ±0.23 8.31 ±0.10
8.51±0.15
7.97 ±0.34
-0.08 ± 0.22
0.45 ±0.47
0.28 ±0.22
0.07 ±0.24
0.62 ±0.36
22 ± 3 °C; 40 ± 15% RH
1
6
21 2.25 x 108
63
99
8.48 ±0.23
8.01 ±0.13
8.59 ±0.23 7.01 ±0.16
4.92 ±1.21
2.69 ±0.91
0.11 ±0.29
0.58 ±0.23
1.57 ±0.25
3.67 ±1.08
5. 90 ±0.82
35 ± 3 °C; 65 ± 15% RH
1
6
21 2.25 x 108
63
99
1.82 ±0.89
0.00 ±0.00
8.59 ±0.23 0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
6.76 ±0.80
8.59 ±0.20
8.59 ±0.20
8.59 ±0.20
8.59 ±0.20
*Data are expressed as mean log survivorship ± standard deviation.
tPositive control coupons were inoculated and extracted at time zero (i.e., one hour after inoculation); one set of positive controls
was used for all test durations.
*Test coupons were inoculated and exposed to the environmental condition for the exposure duration.
§Data are expressed as mean log reduction ± 95% CI.
i/3
01
Low Temp (5 °C / 30% RH)
-Glass
(Non-
Lyophlized)
•Glass
(Lyophilized)
10
20
30
40 50 60
Duration (Days)
70
80
90
100
Figure 4-1. F. pestis Persistence at 5 ± 2 °C and 30 ±15% RH
Data are expressed as mean log survivorship.
12
-------
Moderate Temp (22 °C / 40% RH)
- Glass
(Non-
Lyophlized)
• Glass
(Lyophilized)
10
20
30 40 50 60
Duration (Days)
70
80
90
100
Figure 4-2. Y. pestis Persistence at 22 ±2 °C and 40 ± 15% RH
Data are expressed as mean log survivorship.
High Temp (35 °C / 65% RH)
0.
IE
10
- Glass
(Non-
Lyophlized)
• Glass
(Lyophilized)
10 20 30 40 50 60
Duration (Days)
70
80
90
100
Figure 4-3. Y. pestis Persistence at 35 ± 2 °C and 65 ± 15% RH
Data are expressed as mean log survivorship.
13
-------
Table 4-4. Qualitative Y. pestis Persistence in Soil
Non-Lyophilized Lyophilized
Duration (Days) Inoculum Inoculum
Detected
5 ± 3 °C; 30 ± 15% RH
1 •/
6 S
21
63 S
99 S
^
22 ±3 °C; 40 ± 15% RH
1
6
21
63
99
ND
ND
ND
ND
35 ± 3 °C; 65 ± 15% RH
1
6
21
63
99
ND
ND
ND
ND
ND
v^ = Y. pestis was qualitatively detected in at least one replicate.
ND = Y. pestis was not detected in any replicate.
= samples not tested at that inoculum/time point combination.
4.2.3 F. tularensis
The results obtained for F. tularensis persistence on glass are summarized in Tables 4-5 and 4-6
and Figures 4-4 through 4-6. The qualitative persistence in soil is summarized in Table 4-7. When
lyophilized, F. tularensis persisted on glass for at least 99 days at both low and moderate
temperature and RH conditions (>7 logs and >6 logic recovery, respectively). When not
lyophilized, F. tularensis persisted for at least 99 days at the low temperature/RH conditions but
with lower recoveries (4.62 logic recovery). For the moderate temperature/RH condition, the non-
lyophilized F. tularensis was recoverable after 63 days. Additionally, no F. tularensis was
recovered from glass after 21 days for non-lyophilized or 6 day for lyophilized preparations, at the
high temperature/RH condition.
F. tularensis persisted in soil up to 99 days at the low temperature/RH condition with both sample
preparations. At the moderate temperature/RH condition, F. tularensis persisted longer when
lyophilized compared to non-lyophilized in soil (>63 vs >21 days, respectively). No organisms
were detected after one day in soil at the high temperature/RH condition.
14
-------
Table 4-5. F. tularensis Persistence on Glass Using a Non-Lyophilized Inoculum
Duration
(Days)
Inoculation
Control
(CFU/coupon)
Mean Recovered F. tularensis,
Log Survivorship*
Positive Control Test Coupon*
Mean Log
Reduction8
5 ± 3 °C; 30 ± 15% RH
1
6
21
63
99
1.47 x 108
7.93 ±0.06
7.16 ±0.29
6.93 ±0.43 6.75 ±0.11
5. 10 ±0.25
4.62 ±0.40
-1.00 ±0.38
-0.23 ± 0.46
0.18 ±0.39
1.83 ±0.44
2.31 ±0.52
22 ±3 °C; 40 ± 15% RH
1
6
21
63
99
1.47 x 108
7.24 ±0.08
4.74 ±0.16
6.93 ±0.43 2.34 ±0.32
0.16 ±0.37
0.00 ±0.00
-0.31 ±0.39
2.19 ±0.41
4.59 ±0.47
6.76 ±0.50
6.93 ±0.38
35 ± 3 °C; 65 ± 15% RH
1
6
21
1.47 x 108
1.16 ±0.86
6.93 ±0.43 0.16 ±0.37
0.00 ±0.00
5.76 ±0.84
6.76 ±0.50
6.93 ±0.38
*Data are expressed as mean log survivorship ± standard deviation.
^Positive control coupons were inoculated and extracted at time zero (i.e., one hour after inoculation); one set of positive controls
was used for all test durations.
*Test coupons were inoculated and exposed to the environmental condition for the exposure duration.
§Data are expressed as mean log reduction ± 95% CI.
Table 4-6. F. tularensis Persistence on Glass Using a Lyophilized Inoculum
Inoculation
Duration „ , ,
,_ , Control
(CFU/coupon)
Mean Recovered F. tularensis,
Log Survivorship*
Positive Control Test Coupon*
^H
5 ± 3 °C; 30 ± 15% RH
1
6
21 2.87 x 108
63
99
7.63 ±0.20
7.48 ±0.19
7.40 ±0.27 7.52 ±0.06
7.29 ±0.32
7.63 ±0.19
-0.23 ±0.30
-0.08 ± 0.29
-0.12 ±0.24
0.11±0.37
-0.23 ± 0.29
22 ±3 °C; 40 ± 15% RH
1
6
21 2.87 x 108
63
99
7.27 ±0.22
6.95 ±0.14
7.40 ±0.27 6.51 ±0.39
6.82 ±0.23
6.02 ±0.69
0.13±0.31
0.45 ±0.27
0.90 ±0.42
0.58 ±0.31
1.38 ±0.65
35 ± 3 °C; 65 ± 15% RH
\ 2.87 x 108
6
4.99 ±0.13
7-40±0'27 o.oo ±0.00
2.41 ±0.26
7.40 ±0.24
*Data are expressed as mean log survivorship ± standard deviation.
tPositive control coupons were inoculated and extracted at time zero (i.e., one hour after inoculation); one set of positive controls
was used for all test durations.
*Test coupons were inoculated and exposed to the environmental condition for the exposure duration.
§Data are expressed as mean log reduction ± 95% CI.
15
-------
Low Temp (5 °C / 30% RH)
• Glass
(Non-
Lyophilized)
•Glass
(Lyophilized)
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96
Duration (Days)
Figure 4-4. F. tularemis Persistence at 5 ± 2 °C and 30 ± 15% RH
Data are expressed as mean log survivorship.
Moderate Temp (22 °C / 40% RH)
=
cc
on
•Glass
(Non-
Lyophilized)
•Glass
(Lyophilized)
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96
Duration (Days)
Figure 4-5. F. tularemis Persistence at 22 ± 2 °C and 40 ± 15% RH
Data are expressed as mean log survivorship.
16
-------
High Temp (35 °C / 65% RH)
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96
Duration (Days)
Figure 4-6. F. tularemis Persistence at 35 ± 2 °C and 65 ± 15% RH
Data are expressed as mean log survivorship.
Table 4-7. Qualitative F. tularensis Persistence in Soil
• Glass
(Non-
Lyophilized)
• Glass
(Lyophilized)
Duration (Days)
Inoculum
Lyophilized
Inoculum
5 ± 3 °C; 30 ± 15% RH
1 •/ •/
6 S S
21
-------
days at both low and moderate temperature and RH conditions (>7 logs and >5 logic recovery,
respectively), when lyophilized (Table 4-9). When not lyophilized (Table 4-8), B. mallei persisted
for at least 99 days at the low temperature/RH condition (0.10 logio recovery) and at least six days
at the moderate temperature/RH condition (0.36 logio recovery). No B. mallei was recovered from
glass after six days for either sample preparation at the high temperature/RH condition.
B. mallei persisted in soil up to 99 days at the low temperature/RH condition and up to six days at
the moderate temperature/RH condition with both sample preparations. No organisms were
detected in soil after at any time point/preparation combination at the high temperature/RH
condition.
Table 4-8. B. mallei Persistence on Glass Using a Non-Lyophilized Inoculum
„ ,. Inoculation
Duration „ , ,
~ Control
Clean Recovered B. mallei,
Log Survivorship*
^ontrol1^ Test Coupon*
5 ± 3 °C; 30 ± 15% RH
1
6
21 1.16xl08
63
99
5. 82 ±0.07
5. 93 ±0.12
8.06 ±0.03 4.77 ±0.12
1.74 ±0.92
0.10 ±0.23
2.23 ±0.06
2.13±0.11
3.29±0.11
6.31 ±0.81
7.95 ±0.21
22 ±3 °C; 40 ± 15% RH
1
6
21 1.16xl08
63
99
5. 92 ±0.10
0.36 ±0.57
8.06 ±0.03 0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
2.14 ±0.09
7.69 ±0.50
8.06 ±0.03
8.06 ±0.03
8.06 ±0.03
35 ± 3 °C; 65 ± 15% RH
\ 1.16 xlO8
6
8»6±w» »™
4.87 ±0.31
8.06 ±0.03
*Data are expressed as mean log survivorship ± standard deviation.
^Positive control coupons were inoculated and extracted at time zero (i.e., one hour after inoculation); one set of positive controls
was used for all test durations.
*Test coupons were inoculated and exposed to the environmental condition for the exposure duration.
§Data are expressed as mean log reduction ± 95% CI.
18
-------
Table 4-9. B. mallei Persistence on Glass Using a Lyophilized Inoculum
„ ,. Inoculation
Duration „ , ,
~ , Control
( ys) (CFU/coupon)
Log Survivorship*
Positive Control Test Coupon*
Mean Log
Reduction8
5 ± 3 °C; 30 ± 15% RH
1
6
21 9.60 x 107
63
99
7.41 ±0.14
7.51 ±0.05
7.70 ±0.05 7.27 ±0.11
7.12±0.10
7. 16 ±0.06
0.29 ±0.13
0.18 ±0.06
0.42 ±0.11
0.57 ±0.10
0.53 ±0.07
22 ±3 °C; 40 ± 15% RH
1
6
21 9.60 x 10'
63
99
6.63 ±0.61
5. 91 ±0.94
7.70 ±0.05 6.41 ±0.18
6.01 ±0.02
5. 53 ±0.28
1.07 ±0.53
1.78 ±0.83
1.29 ±0.17
1.68 ±0.05
2.16 ±0.25
35 ± 3 °C; 65 ± 15% RH
\ 9.60 x 107
6
5.64 ±0.94
7-70±0-05 o.oo ±0.00
2.06 ±0.83
7.70 ±0.04
*Data are expressed as mean log survivorship ± standard deviation.
tPositive control coupons were inoculated and extracted at time zero (i.e., one hour after inoculation); one set of positive controls
was used for all test durations.
*Test coupons were inoculated and exposed to the environmental condition for the exposure duration.
§Data are expressed as mean log reduction ± 95% CI.
Low Temp (5 °C / 30% RH)
I
VI
• Glass
(Non-
Lyophilized)
•Glass
(Lyophilized)
10
20
30
40 50 60
Duration (Days)
70
80
90
100
Figure 4-7. B. mallei Persistence at 5 ± 2 °C and 30 ± 15% RH
Data are expressed as mean log survivorship.
19
-------
Moderate Temp (22 °C / 40% RH)
- Glass
(Non-
Lyophilized)
•Glass
(Lyophilized)
10
20
30 40 50 60
Duration (Days)
70
80
90
100
Figure 4-8. B. mallei Persistence at 22 ± 2 °C and 40 ± 15% RH
Data are expressed as mean log survivorship.
High Temp (35 °C / 65% RH)
-Glass
(Non-
Lyophilized)
• Glass
(Lyophilized)
20 30 40 50 60
Duration (Days)
70
80
90
100
Figure 4-9. B. mallei Persistence at 35 ± 2 °C and 65 ± 15% RH
Data are expressed as mean log survivorship.
20
-------
Table 4-10. Qualitative B. mallei Persistence in Soil
Non-Lyophilized
Inoculum
Detected
Lyophilized
Inoculum
5 ± 3 °C; 30 ± 15% RH
1 •/ •/
6 S S
21 S S
63 S S
99 S S
22 ±3 °C; 40 ± 15% RH
1
6
21
63
99
1
6
21
63
99
ND
ND
ND
35 ± 3 °C; 65 ± 15% RH
ND
ND
ND
ND
ND
ND
ND
S = B. mallei was qualitatively detected in at least one replicate.
ND = B. mallei was not detected in any replicate.
= samples not tested at that inoculum/time point combination.
21
-------
5.0 Summary
When exposed to 5 ± 3 °C and 30 ± 15% RH, 7 pestis, F. tularensis, and B. mallei all persisted
on glass at a higher degree when lyophilized rather than non-lyophilized. Once lyophilized, little
to no reduction in viability was observed on glass over the 99 days. In comparison, organisms from
non-lyophilized samples were still recoverable, but to a lesser degree (logic recovery ranged from
0.10 to 4.62). Although not quantitatively evaluated, organisms were also detected in soil up to 99
days at the low temperature/RH combination for all three organisms tested, regardless of the
preparation.
When exposed to 22 ± 3 °C and 40 ± 15% RH, all organisms again persisted on glass at a higher
degree when lyophilized rather than air dried. Once lyophilized, 2.69 (7. pestis), 6.02 (F.
tularensis), and 5.53 (B. mallei) logic recovery was obtained after a 99-day contact time. When
not lyophilized, no organisms were recovered after six days for 7 pestis and B. mallei, or after 63
days for F. tularensis. In soil, the same pattern was observed with organisms generally persisting
at least as long, if not longer when lyophilized (7 pestis: six days for both preparations; F.
tularensis: 63 days versus 21 days; B. mallei: six days for both preparations).
Lastly, all organisms were inactivated by exposure to 35 ± 3 °C and 65 ± 15% RH sooner than the
low and moderate temperature/RH conditions, regardless of inoculation method. No 7 pestis was
recovered from glass after one day using the non-lyophilized inoculum or after six days using the
lyophilized inoculum (1.82 logic recovery after one day). No F. tularensis was recovered from
glass after 21 days for the non-lyophilized preparations (0.16 logic recovery after six days) or after
six days using the lyophilized inoculum (4.99 logic recovery after one day). Lastly, no B. mallei
was recovered from glass after six days for either preparation technique (3.19 and 5.64 logs, for
non-lyophilized, respectively). No 7 pestis or F. tularensis was detected in soil after six days, and
no B. mallei was detected after one day when not lyophilized. When lyophilized, 7 pestis was
detected out to six days, F. tularensis out to one day, and no B. mallei was detected after only one
day.
The minimum exposure time where no organisms were recovered on either glass or soil are
summarized in Table 5-1.
Table 5-1. Minimum Exposure Times for Organism Recovery on Glass or Soil
.... , Lyophilized
Non- T , ... ,
.... , Lyophilized
Non- T , ... ,
.... , Lyophilized
5 ± 3 °C; 30 ± 15% RH
>99 days
>99 days
>99 days >99 days
>99 days
>99 days
22 ± 3 °C; 40 ± 15% RH
>6 days
>99 days
>63 days >99 days
>6 days
>99 days
35 ± 3 °C; 65 ± 15% RH
>lday
>6 days
>6 days >1 day
>lday
>lday
These data suggest that temperature and RH, as well as the preparation of the organism,
significantly impact the persistence of 7 pestis, F. tularensis, and B. mallei on glass and in soil.
All three organisms will apparently survive for extended periods of time (potentially greater than
99 days) when lyophilized and released into the environment, which may warrant additional
decontamination investigations. However, the use of elevated temperature may be a viable
22
-------
decontaminant option for some situations, as all three organisms were inactivated to a point of
non-detect after six days in most instances (lyophilized Y. pestis was detected in soil after six days
at 35 °C and 65% RH). These persistence data are useful for making response or remediation
decisions following natural occurrences or intentional releases of biological agents.
23
-------
6.0 References
1. ASTM El04. Standard Practice for Maintaining Constant Relative Humidity by Means of
Aqueous Solutions. ASTM International, 2012.
2. Heckly RJ, Blank H. Virulence and viability of Yersiniapestis 25 years after lyophilization.
Applied and Environmental Microbiology. 1980;3 9(3): 541-543.
3. http://opsdiagnostics.com/applicati ons/lyophilization/ecoli_lyophilizati on_stability.html,
last accessed October 16, 2015.
24
-------
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
------- |