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600/R-11/111
October 2011
Technology Evaluation Report
Persistence and Decontamination
Testing of Bruce/fa suis
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
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Notice
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's National Homeland Security Research Center, funded, directed and managed this
work through Contract Number EP-C-10-001 with Battelle. This report has been peer and
administratively reviewed and has been approved for publication as an EPA document. Note that
approval does not signify that the contents necessarily reflect the views of the agency.
Mention of trade names or commercial products does not constitute endorsement or
recommendation for use of a specific product.
Questions or comments should be addressed to:
M. Worth Calfee
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
in
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Acknowledgments
Contributions of the following individuals and organizations to the development of this
document are acknowledged.
United States Environmental Protection Agency (EPA)
Office of Research and Development, National Homeland Security Research
Center
Jeff Szabo
Office of Emergency Management
Michael E. Ottlinger
Office of Chemical Safety and Pollution Prevention
Rebecca Pines
Battelle Memorial Institute
IV
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Foreword
Following the events of September 11, 2001, addressing the critical needs related to homeland security
became a clear requirement with respect to EPA's mission to protect human health and the environment.
Presidential Directives further emphasized EPA as the primary federal agency responsible for the country's
water supplies and for decontamination following a chemical, biological, and/or radiological (CBR) attack.
To support the EPA mission with respect to response and recovery from incidents of national significance,
the National Homeland Security Research Center (NHSRC) was established to conduct research and
deliver products that improve the capability of the Agency to carry out its homeland security
responsibilities.
One specific goal of NHSRC's research is to provide information on decontamination methods and
technologies that can be used in the response and recovery efforts resulting from a CBR contamination
event. In recovering from an event, it is critical to identify and implement decontamination technologies
that are appropriate for the given situation. In a wide-area attack scenario, the decontamination approach
must be effective; while at the same time must be readily available, and easily deployed. The fate and
persistence of the agent must also be considered prior to developing a decontamination strategy.
The current study investigated the persistence of Brucella suis, a vegetative bacterial threat agent, on
common building materials at two environmental conditions. In addition, several readily-available liquid
decontaminants were evaluated for their ability to inactivate B. suis on surfaces. Information on the
effectiveness of these technologies is provided to inform both decontaminant selection and implementation.
These results, coupled with additional information in separate NHSRC publications (available at
www.epa. gov/nhsrcX can be used to determine whether a particular decontamination technology can be
effective in a given scenario. With these factors in consideration, the best technology or combination of
technologies can be chosen that meets the clean up, cost and time goals for a particular decontamination
scenario.
NHSRC has made this publication available to assist the response community prepare for and recover from
disasters involving biological contamination. This research is intended to move EPA one step closer to
achieving its homeland security goals and its overall mission of protecting human health and the
environment while providing sustainable solutions to our environmental problems.
- Jonathan Herrmann, P.E., Director
National Homeland Security Research Center
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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. The generation of scientifically
defensible persistence data is useful for the proper planning of decontamination efficacy tests and
for formulating 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 ofBmcella suis (B. suls) on three material types (typically found in
buildings) under multiple environmental conditions and time points. The efficacy of three liquid
decontaminants against B. suis was also investigated on the same three materials.
Persistence (recovery of viable organisms) was assessed for B. suis inoculated onto three
materials (aluminum, glass, and wood) maintained under controlled environmental conditions for
up to 56 days. The environmental conditions consisted of moderate temperature and relative
humidity (22 °C ± 2 °C; 40% RH ± 15%) or low temperature and relative humidity (5 °C ± 3 °C;
30% RH ±15%). B. suis persisted on all material types at all time points and under both sets of
environmental conditions. The only exception was the inability to recover viable B. suis on wood
after 56 days at moderate temperature and relative humidity. A higher log survivorship
(exhibited increased persistence) was observed for B. suis at low temperature and relative
humidity than at moderate temperature and relative humidity. The persistence testing results are
summarized in Table ES-1.
Table ES-1. Summary of Persistence Testing
Material TemPecrature' % RHt
Mean recovered/?, suis (log survivorship) by Duration in Days*§
0 14 28 42 56
22 ±2 °C; 40 ± 15% RH
Aluminum
Glass
Wood
Aluminum
Glass
Wood
22.5
22.5
22.4
6.4
5.7
5.9
43.7
44.0
44.7
33.6
34.6
33.9
8.63
8.60
7.59
5±3
8.63
8.60
7.59
3.99
3.81
4.18
°C; 30 ± 15% RH
7.12
7.07
6.14
3.45
3.20
0.53
6.70
6.64
5.77
2.68
3.06
0.40
6.48
6.74
5.68
2.51
2.78
ND
6.35
6.25
2.67
*Data are expressed as mean logs recovered.
t Mean temperature and RH values based on continuous monitoring at 6 minute intervals over the entire 56 days.
"ND" indicates that no viable organisms were recovered from any of the replicate coupons.
^Starting inoculum was 8.92 logs.
The efficacy of three decontaminants (pH-amended Ultra Clorox® bleach, 70% ethanol [EtOH],
and 1% citric acid) was also assessed at two time points (0 and 28 days) under both sets of
environmental conditions. Efficacy was reported as reduction due to attenuation (not persisting),
reduction due to decontamination, and combined reduction for each of the three decontaminants
tested. The reduction due to attenuation refers to the log reduction (as calculated by Equation 2)
of persistence test coupons held at their respective environmental conditions for 28 days. The
reduction due to decontamination refers to the log reduction of B. suis due to the decontaminant
(and not the attenuation of the organism on the coupons) when compared to the efficacy of the 28
day persistence test coupons instead of the positive controls (time 0). The combined reduction
VI
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refers to the overall log reduction of the samples due to attenuation in addition to
decontamination.
All three decontaminants were more effective (exhibited higher log reductions) on the nonporous
aluminum and glass compared to porous wood. Also, pH-amended Ultra Clorox® bleach and
70% EtOH were more effective against B. suis on all three materials types than 1% citric acid.
Interestingly, at low temperature, 70% EtOH demonstrated higher efficacy against B. suis on
wood than pH-amended Ultra Clorox® bleach. These data suggest that environmental conditions
and material type should be considered when choosing a decontaminant. Also, some
disinfectants (e.g., 1% Citric Acid) demonstrate poor efficacy against B. suis on environmental
surfaces. The efficacy results for all three decontaminants are summarized in Tables ES-2, ES-3
and ES-4.
Table ES-2. Summary of Brucella suis Reduction by Decontamination and Attenuation
Over Time on Test Materials for pH-Amended Ultra Clorox® Bleach
Test Material
Quantitative Efficacy (log reduction)*
Duration, Days Reduction due Reduction due Combined
to Attenuation to Decon Reduction+
22±2°C;40±15%RH
Aluminum
Glass
Wood
0
28
0
28
0
28
5.18
5.40
7.05
>8.54
>3.45
>8.43
>3.20
1.22
>0.53
>8.63
>8.60
>7.59
5±3°C;30±15%RH
Aluminum
Glass
Wood
0
28
0
28
0
28
1.94
1.95
1.81
>8.54
>6.70
>8.43
>6.64
1.22
0.51
>8.63
>8.60
2.33
*Results presented as mean log reduction.
Log reduction due to attenuation and decontamination of B. suis after 28
days.
Vll
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Table ES-3. Summary of Brucella suis Reduction by Decontamination and Attenuation on
Test Materials for 70% Ethanol
Test Material
Durati
Day:
Quantitative
on,
s Reduction due
to Attenuation
Efficacy (log reduction)*
Reduction due
to Decon
Combined
Reduction"1"
22±2°C;40±15%RH
Aluminum
Glass
Wood
0
28
0
28
0
28
5.18
5.40
7.05
>8.54
>3.45
>8.43
>3.20
2.15
0.13
>8.63
>8.60
7.19
5±3°C;30±15%RH
Aluminum
Glass
Wood
0
28
0
28
0
28
1.94
1.95
1.81
>8.54
>6.70
>8.43
>6.64
2.15
3.26
>8.63
>8.60
5.07
*Results presented as mean log reduction.
Log reduction due to attenuation and decontamination of B. suis after 28 days.
Table ES-4. Summary of Brucella suis Reduction by Decontamination and Attenuation on
Test Materials for 1% Citric Acid
Test Material
Duration,
Days
Quantitative Efficacy (log reduction)*
Reduction _ , ,. ,
, , Reduction due
due to _
. , , , . to Decon
Attenuation
Combined
Reduction+
22±2°C;40±15%RH
Aluminum
Glass
Wood
Aluminum
Glass
Wood
0
28
0
28
0
28
5±3
0
28
0
28
0
28
5.18
5.40
7.05
°C;30±15%RH
1.94
1.95
1.81
1.78
>3.45
1.30
>3.20
0.76
-0.29
1.78
5.69
1.30
3.75
0.76
0.38
>8.63
>8.60
6.76
7.62
5.70
2.19
*Results presented as mean log reduction.
+ Log reduction due to attenuation and decontamination of B. suis after 28 days.
Vlll
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Contents
Notice iii
Acknowledgments iv
Foreword v
Executive Summary vi
Abbreviations/Acronyms xii
1.0 Introduction 1
2.0 Procedures 2
2.1 Biological Agent 2
2.2 Test Materials 2
2.3 Coupon Inoculation 3
2.4 Environmental Conditions and Persistence Testing Procedure 4
2.5 Decontamination Procedure 5
2.6 B. suis Recovery 5
3.0 Quality Assurance/Quality Control 8
3.1 Performance Evaluation Audit 8
3.2 Technical Systems Audit 8
3.3 Data Quality Audit 8
3.4 QA/QC Reporting 8
3.5 Data Review 9
3.6 Performance Criteria 9
3.7 Deviation 9
4.0 Persistence Test Results 10
4.1 Method Demonstration - B. suis Recovery 10
4.2 Persistence Results 10
4.2.1 Aluminum 10
4.2.2 Glass 11
4.2.3 Wood 12
5.0 Decontamination Test Results 14
5.1 pH-Amended Ultra Clorox® 14
5.1.1 Neutralization Methodology 14
5.1.2 pH-amended Ultra Clorox® Test Results 15
5.2 70%Ethanol 17
5.2.1 Neutralization Methodology 17
5.2.2 70% Ethanol Test Results 18
5.3 1% Citric Acid 20
5.3.1 Neutralization Methodology 20
5.3.2 1% Citric Acid Test Results 21
5.4 Surface Damage to Materials 23
6.0 Summary 24
7.0 References 25
Appendix A 26
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Figures
Table ES-1. Summary of Persistence Testing vi
Table ES-2. Summary of Brucella suis Reduction by Decontamination and Attenuation Over
Time on Test Materials for pH-Amended Ultra Clorox® vii
Table ES-3. Summary of'Brucella suis Reduction by Decontamination and Attenuation on Test
Materials for 70% Ethanol viii
Table ES-4. Summary of Brucella suis Reduction by Decontamination and Attenuation on Test
Materials for 1% Citric Acid viii
Figure 2-1. Inoculation of coupon using a multichannel pipette 3
Figure 4-1. B. suis persistence at 22 °C ± 2 °C and 40% RH ± 15% 12
Figure 4-2. B. suis persistence at 5 °C ± 3 °C and 30% RH ± 15% 13
Figure 5-1. Inactivation of B. suis using pH-amended Ultra Clorox® 17
Figure 5-2. Inactivation of B. suis using 70% ethanol 20
Figure 5-3. Inactivation of B. suis using 1% citric acid 23
Tables
Table 2-1. Test Materials 3
Table 2-2. Summary of Temperature and Relative Humidity Conditions During Persistence
Testing 4
Table 3-1. Performance Evaluation Audits 8
Table 3 -2. Persistence Testing Inoculation and Positive Control Recovery Data 9
Table 4-1. Recovery Data from Method Demonstration 10
Table 4-2. B. suis Persistence on Aluminum 11
Table 4-3. B. suis Persistence on Glass 11
Table 4-4. B. suis Persistence on Wood 12
Table 5-1. Spray-and-Weigh for pH-Amended Ultra Clorox® Deposition on Three Outdoor
Materials 14
Table 5-2. Neutralization Testing for pH-Amended Ultra Clorox® with B. suis Against
Glass and Wood Test Materials 14
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Table 5-3.
Table 5-4.
Table 5-5.
Table 5-6.
Table 5-7.
Table 5-8.
Table 5-9.
Neutralization Testing for pH-Amended Ultra Clorox with B. suis Against
Aluminum Test Materials 15
Inactivation of B. suis using pH-Amended Ultra Clorox® 16
Spray-and-Weigh for 70% Ethanol Deposition on Three Outdoor Materials 17
Neutralization Testing for 70% Ethanol with B. suis Against Aluminum, Glass, and
Wood Test Materials 17
Inactivation of B. suis using 70% Ethanol 19
Spray-and-Weigh for 1% Citric Acid Deposition on Three Outdoor Materials 20
Neutralization Testing for 1% Citric Acid with B. suis Against Aluminum, Glass,
and Wood Test Materials 21
Table 5-10. Inactivation of B. suis using 1% Citric Acid 22
XI
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Abbreviations/Acronyms
ATCC
BHI
BSC
CPU
CI
EPA
D/E
EtOH
mL
NHSRC
NIST
OD
ORD
PBS
ppm
QA
QC
QMP
RH
SE
SFW
STS
TSA
T&E
WA
American Type Culture Collection
brain heart infusion
biosafety cabinet
colony-forming unit(s)
confidence interval
U.S. Environmental Protection Agency
Dey/Engley
ethanol
milliliter
National Homeland Security Research Center
National Institute of Standards and Technology
optical density
Office of Research and Development
phosphate-buffered saline
parts per million
quality assurance
quality control
Quality Management Plan
relative humidity
standard error
sterile filtered water (cell-culture grade)
sodium thiosulfate
technical systems audit
Testing and Evaluation
work assignment
xn
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1.0 Introduction
The U.S. Environmental Protection Agency's
(EPA's) National Homeland Security
Research Center (NHSRC) is helping protect
human health and the environment from
adverse impacts resulting from acts of terror.
The U.S. EPA is investigating the
persistence of biological and chemical
agents in the absence of decontamination.
For biological agents, persistence reflects
the extent to which viability is retained over
defined periods of time. Some biological
agents are unstable and lose viability within
minutes of their release, thereby diminishing
the risk to human health and the
environment; other agents can remain viable
for weeks, months, or years. The
persistence of biological agents is influenced
by environmental conditions and the
materials with which they are in contact.
The generation of scientifically defensible
persistence data is useful for the proper
planning of decontamination efficacy tests
and helps formulate response plans in
preparation for possible natural occurrences
or intentional releases of biological agents.
Once persistence of biological agents is
assessed, the emphasis focuses on
decontamination and consequence
management and threat and consequence
assessment. NHSRC is working to develop
tools and information that will help detect the
intentional introduction of chemical or
biological contaminants into building systems,
contain these contaminants, decontaminate
buildings and facilitate disposal of the material
resulting from cleanups.
This investigation focused on the persistence
and decontamination of Brucella suis, a
bacterial select agent that can cause a
debilitating influenza-like illness in humans.
The intent was to determine the length of
time that B. suis remained viable on various
materials found in buildings (aluminum,
glass, and wood) under two sets of
environmental conditions. One
environmental condition approximated
temperature and humidity expected in a
typical climate-controlled indoor
environment, and one environmental
condition approximated conditions known to
enhance B. suis survivorship (low
temperature and humidity). Material
coupons were also decontaminated with
three liquid decontaminants (pH-amended
Ultra Clorox®, 70% ethanol, and 1% citric
acid) after being exposed to two
environmental conditions for two lengths of
time to determine the efficacy of these
decontaminants. These decontaminants
were chosen based upon their expected high
availability following a bioterror incident.
Two are disinfectants (ethanol and citric
acid), and one is a sporicidal decontaminant
(pH-amended Ultra Clorox® bleach). Data
presented herein may be used to guide
decontaminant selection during cleanup
activities following a biological attack with
Brucella.
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2.0 Procedures
This report describes the investigation of the
persistence and decontamination of B. suis
on three different materials exposed to two
environmental conditions for various lengths
of time. Briefly, materials (aluminum, glass,
and wood) were inoculated with B. suis (~1
x 109 colony forming units/milliliter
[CFU/mL]) and then exposed to two
environmental conditions (22 °C ± 2 °C and
40% RH ± 15% or 5 °C ± 3 °C and 30% RH
± 15%) for controlled exposure durations up
to 56 days. Persistence was determined for
each material and environmental condition
at 0, 14, 28, 42, and 56 days. Additional
replicates of these materials were also
decontaminated at 0 and 28 days using pH-
amended Ultra Clorox® bleach, 70% EtOH,
or 1% citric acid. Persistence and
decontamination were assessed by
measuring the recovery of B. suis as CPU
from each tested combination of material,
environmental condition, exposure duration,
and/or decontaminant. All testing was
performed in accordance with the peer-
reviewed and EPA-approved Quality
Assurance/Test Plan for Persistence Testing
ofBrucella suis on Outdoor Materials,
October, 2008,(1) and associated
amendments.
2.1 Biological Agent
The biological agent B. suis is a gram-
negative, aerobic, non-spore-forming
coccobacillus.(2) B. suis biotype I (Battelle
BRU163) was obtained from American
Type Culture Collection (ATCC - Manassas,
VA) in January, 2004 and maintained in
pure culture by Battelle for this
investigation. The stock culture of B. suis
was identified by whole genome sequencing
and confirmed to be Brucella suis 1330
(ATCC23444), a fully-virulent strain. This
procedure and results are described in
Appendix A.
A fresh B. suis culture was prepared in
advance of each day that coupons were
inoculated by transferring colonies from a
streak plate (freshly growing or stored less
than two weeks at 2 °C to 8 °C) into 10 mL
of Brain Heart Infusion (BHI) broth (BD
Diagnostic Systems, Sparks, MD). This
culture was then incubated overnight at 37
°C ± 2 °C on an orbital shaker set to 200
revolutions per minute, until an increase in
turbidity was observed. The bacterial
culture (late log phase of growth) was
diluted with BHI broth to an optical density
(OD) reading at 600 nm (OD600 nm) of
approximately 0.1 to 0.2 OD units. The
viable B. suis bacteria in the stock
suspension were enumerated to determine
CFU/mL ("inoculation control") by
analyzing serial dilutions (serial 1:10
dilutions) of the stock suspension prepared
using phosphate-buffered saline (PBS) and
plated onto BHI agar (BD Diagnostic
Systems, Sparks, MD) for CPU
determination.
2.2 Test Materials
Materials typically found within built
environments that were used for this study
are described in Table 2-1. Test coupons of
these materials were cut to the sizes
indicated in Table 2-1 from larger pieces of
stock material. All coupons were
constructed of new, unsoiled materials.
Coupons were sterilized either by
autoclaving or by gamma irradiation. The
selected approach, as shown in Table 2-1,
was based on cost-effectiveness and
minimization of physical alterations of the
material. Autoclaving followed an internal
Battelle standard operating procedure, and
gamma-irradiation at 40 kilogray was
conducted by STERIS Isomedix Services
(Libertyville, IL). Prior to gamma
irradiation, coupons were sealed in 6 mil
Uline® poly tubing (Uline, Chicago, IL) to
preserve sterility until the coupons were
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ready for use. Test coupons were each
visually inspected prior to being used in any
test. Coupons with anomalies on the
application surface were discarded and not
used. Blank coupons (not inoculated with B.
suis) confirmed sterility of each material
type.
Table 2-1. Test Materials
Material
Aluminum
(finished)
Glass
Wood
(Untreated Pine)
Lot, Batch, or
Observation
Alloy 2024
C1036
Generic
Molding
Manufacturer or
Supplier Name
Adept Products, Inc.
Columbus, OH
Brooks Brothers
Columbus, OH
Lowes
Columbus, OH
Coupon Size,
Width x Length
1.9 cmx7.5 cm
1.9 cmx7.5 cm
1.9 cmx7.5 cm
Coupon
Thickness
0.2cm
0.3cm
0.5 cm
Material
Preparation
Autoclave
Autoclave
Gamma
Irradiation
2.3 Coupon Inoculation
The stock suspension (approximately 1 x 109
CFU/mL of B. suis, prepared as described in
Section 2.1) was used to inoculate the
coupons. Test and positive control coupons
were placed lying flat in a Class III
biological safety cabinet (BSC) and
inoculated with a 100 uL aliquot of stock
suspension using a multichannel
micropipette as two rows of five droplets
(10 uL per droplet) across the surface of the
coupons (Figure 2-1). This 100 uL aliquot
of stock suspension yielded approximately 1
x 108 CPU/coupon. Inoculated coupons
were allowed to dry for 2 hours prior to
being extracted (positive control coupons),
or exposed to the test conditions or
decontaminants as prescribed in the test
plan. This method of coupon inoculation,
liquid inoculation, offers the high
repeatability necessary for data
comparisons, and may not accurately
simulate all B. suis contamination scenarios.
Figure 2-1. Inoculation of coupon using a multichannel pipette.
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2.4 Environmental Conditions and
Persistence Testing Procedures
Persistence and decontamination testing
were conducted under two sets of
environmental conditions:
Moderate temperature and relative
humidity; 22 °C ± 2 °C and 40%
RH± 15%
Low temperature and relative
humidity; 5 °C ± 3 °C; 30% RH ±
15%
For all persistence testing and prior to the
Day 28 decontamination test, the test
coupons were held at the required
temperature for the required time period (up
to 56 days) in an airtight persistence testing
chamber (Lock & Lock, HPL838P, Farmers
Branch, TX). After the coupons were
removed from their respective persistence
testing chambers, all were moved to a
decontamination testing chamber (BSC III)
for application of the decontaminant or to a
BSC II for extraction (persistence test
coupons only).
The chambers containing the low
temperature and relative humidity (5 °C ± 3
°C; 30% RH ± 15%) persistence test
coupons were kept in a refrigerator and
magnesium chloride hexahydrate
(MgCl2.6H2O) was added to maintain the
relative humidity in the chambers. The
chambers containing the moderate
temperature and relative humidity (22 °C ±
2 °C; 40% RH ± 15%) test coupons were
kept at ambient laboratory temperature and
potassium carbonate (K2CO3) was added to
maintain the relative humidity in the
chambers. Both fixed humidity point salts
(MgCl2.6H2O and K2CO3) were mixed with
water and added as a slurry to separate
containers and placed in the bottom of the
appropriate persistent test chambers.1-3-1 The
coupons were never allowed to come into
direct contact with the slurry or the slurry
containers. The temperature and relative
humidity for both conditions were recorded
approximately every 6 minutes using a
HOBO data logger (Onset Computer
Corporation, Bourne, MA). The actual
temperatures and relative humidity levels
observed during testing are documented in
Table 2-2. A rise and/or fall of temperature
and relative humidity was seen when
extracting coupons from the persistence
testing chambers at their respective time
points. However, all coupons returned to the
appropriate target testing condition within
12 hours 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.
Table 2-2. Summary of Temperature and Relative Humidity Conditions During Persistence
Testing*f
Target Testing
Condition
22 °C ± 2 °C;
40%RH±15%
5 °C ± 3 °C;
30%RH±15%
Aluminum
Temp.,°C
22.5 ±0
6.4 ±0.
.17
76
%RH
43.7 ±0
33.6 ±1
.31
.45
Temp.,°C
22.5 ±0
5.7 ±0.
.18
73
Glass
%RH
44.0 ± 0.46
34.6 ±0.92
Temp.,°C
22.4 ± 0
5.9±0.
.19
72
Wood
%RH
44.7 ±0.
33.9±1.
,72
,15
*Data are presented as mean ± standard deviation.
t Mean temperature and RH values based on continuous monitoring at 6 minute intervals over the entire 56 days.
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2.5 Decontamination Procedure
All decontamination tests were conducted
under ambient conditions inside a test
chamber (BSC III) in a climate-controlled
laboratory. The test chamber was allowed to
equilibrate to laboratory conditions prior to
decontamination of the coupons. Relative
humidity in the chamber was monitored
using a National Institute of Standards and
Technology (NIST) -traceable hygrometer;
however, no attempt was made to control the
relative humidity in the chamber. The
relative humidity never exceeded 62% in the
decontamination test chamber.
All decontaminants were applied 12 inches
from the horizontally-oriented coupons until
the coupons appeared saturated with liquid.
To simulate field-use conditions, a handheld
sprayer (Cat. 733IX, Qorpak, Bridgeville,
PA) was used to apply the sterile filtered
water (SFW, for positive control samples
only) and decontaminants. Close
observation of the respective coupon
surfaces were made to ensure that they were
thoroughly wetted. The aluminum and glass
coupons were kept in contact with the SFW
or decontaminant for 15 minutes. A re-
application (approximately 2 seconds) of the
SFW decontaminant was done for wood at
15 minutes and it was kept in contact with
the SFW or decontaminant for a total of 30
minutes. After the 15 or 30 minute contact
time, each coupon was kept in its horizontal
position and carefully placed in a 50 mL
conical tube in a manner to avoid loss of any
remaining liquid decontaminant that may
have pooled on the coupon surface (e.g.,
runoff). The conical tubes contained 10 mL
of the appropriate neutralizing buffer for
each decontaminant (refer to Section 5.0) or
10 mL PBS for the positive control coupons.
2.6 B. suis Recovery
Neutralization of test decontaminants during
efficacy testing is necessary to achieve
prescribed contact times and to prevent
downstream effects of residual
decontaminants during culture-based assays.
Neutralization of the pH-amended Ultra
Clorox® bleach was achieved with sodium
thiosulfate (STS). To determine the amount
of pH-amended Ultra Clorox® bleach
applied to the coupons during the spray
procedure, triplicate coupons of each
material were weighed prior to spraying, and
these values were recorded. The materials
were then sprayed with the formulation as
they would be during the decon tests,
allowed a 15 or 30 minute contact time, and
each material was weighed again (wood
required a single re-application during the
30 min time span). The pre-spray values
were subtracted from the post-spray values.
An average value was used to calculate the
amount of decontaminant applied to the
coupon and determine the mass of STS
needed to effectively neutralize the pH-
amended Ultra Clorox® bleach for each
material type. Optimal neutralization
conditions for 70% EtOH and 1% citric acid
were determined similarly, but with 100%
Dey Engley Broth (BD Diagnostic Systems,
Franklin Lakes, NJ) as the neutralizer.
For sample extraction, persistence test
coupons, positive controls, and blanks were
transferred aseptically to sterile individual
50 mL conical vials containing 10 mL of
sterile PBS extraction buffer.
Decontamination test coupons and blanks
were placed in vials containing 10 mL of the
appropriate neutralization buffer. All vials
were agitated on an orbital shaker for 15
minutes at approximately 200 revolutions
per minute at room temperature. Following
extraction from the coupons, the extracts
were removed and a series of dilutions
(serial 1:10 dilutions) was prepared using
PBS. An aliquot (0.1 mL) of the selected
dilutions and, when necessary, the undiluted
extract were plated onto BHI agar in
triplicate. The cultures were incubated for
up to 72 hours at 37 °C ± 2 °C. The colonies
were counted manually and the CFU/mL
determined. Typically, plates having colony
counts between 25 and 250 are used for
calculating the CFU/mL. However, under
certain circumstances (i.e., poor recovery,
reduced persistence over time, efficient
-------
decontamination, etc.) 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 CPU/coupon was calculated by
multiplying the CFU/mL by the volume of
the extraction buffer used for each coupon
(10 mL per coupon). The total CPU
extracted from a coupon were calculated as:
Total CFU/coupon = [(mean CFU plate count x I/dilution factor)/plated volume] x (extraction or
neutralization buffer volume) (1)
where:
Mean CFU plate count
Plated volume
Dilution factor
Extraction/neutralization
buffer volume
= average number of colonies counted on three
replicate plates
= 0.1 mL
= portion of the total extraction buffer that was used
to prepare the dilutions
= 10mL
A single viable bacterium present in a plated
aliquot of sample would be expected to be
observed as a CFU. Therefore, if one CFU
is observed on one of the three plates of
undiluted extract, the individual coupon
detection limit is approximately 33
CFU/coupon based on the above equation.
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 collected for plating. However,
given the number of replicate coupons (five)
and replicate plates (three) per undiluted
coupon extract, there is a low probability
that the presence of viable bacteria would go
undetected.
The recovery of B. suis bacteria (quantified
as mean CFU/coupon ± standard deviation)
was calculated for each
material/environmental condition/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). The
performance or efficacy of each
decontamination technology was assessed
by determining the number of viable
organisms remaining on each test coupon
and in any decontaminant runoff from the
coupon, after decontamination. These data
were compared with the number of viable
organisms extracted from the positive
control coupons sprayed with SFW.
Efficacy was reported as reduction due to
attenuation (not persisting), reduction due to
decontamination, and combined reduction
for each of the three decontaminants tested.
The reduction due to attenuation refers to
the log reduction (as calculated by Equation
2) of persistence test coupons held at their
respective environmental conditions for 28
days. The reduction due to decontamination
refers to the log reduction of B. suis due to
the decontaminant (and not the attenuation
of the organism on the coupons) when
compared to the efficacy of the 28 day
persistence test coupons instead of the
positive controls (time 0). The combined
reduction refers to the overall log reduction
of the samples due to attenuation in addition
to decontamination.
The abundance of viable B. suis in extracts
of test and positive control coupons was
determined to calculate efficacy of the
decontaminant. Efficacy is defined as the
extent (as log reduction) by which viable
cells extracted from test coupons after
decontamination were less numerous than
the viable cells extracted from positive
control coupons subjected only to an inert
SFW spray, at the same temperature and
contact time as the decontaminant
application. First, the logarithm of the CFU
abundance value from each coupon extract
-------
was determined, and then the mean of those
logarithm values was determined for each
set of control and associated test coupons,
respectively. Efficacy of a decontaminant
for a test organism on the /'th coupon material
was calculated as the difference between
those mean log values, i.e.:
Efficacy = (log CFUci}) - (log CFUtt])
(2)
where log CFUCy refers to the 7* individual
logarithm values obtained from the positive
control coupons and log CFUty refers to the
fh individual logarithm values obtained from
the corresponding test coupons, and the
overbar designates a mean value. The only
exception to this equation is for the day 28
decontamination test materials. In this case
log CFUCy refers to the 7* individual
logarithm values obtained from the day 28
persistence test coupons instead of the
positive controls. In tests conducted under
this plan, there were five control and five
corresponding test coupons (i.e.,7 = 5). In
the case where no CPU were found in a
coupon extract, a CPU value of 1 was
assigned, resulting in a log CPU of zero for
that coupon. This situation occurred
frequently when a decontaminant was highly
effective, and no CPU were found in the
plated aliquot of extract from the
decontaminated test coupons. In such cases,
the final efficacy was reported as greater
than or equal to (>) the value calculated by
Equation 2.
The variances (i.e., the square of the
standard deviation) of the log CFUCy and
log CFUty values were also calculated for
both the control and test coupons (i.e., S2cij
and S%), and were used to calculate the
pooled standard error (SE) for the efficacy
value calculated in Equation 2, as follows:
SE =
(3)
where the number 5 again represents the
number/ of coupons in both the control and
test data sets. Thus each efficacy result is
reported as a log reduction value with an
associated SE value.
The significance of differences in efficacy
across different coupon materials and
decontaminant types was assessed based on
the 95% confidence interval of each efficacy
result. The 95% confidence interval (CI) is:
95% CI = Efficacy ± (1.96 x SE)
(4)
-------
3.0 Quality Assurance/Quality Control
Quality assurance (QA)/quality control
procedures were performed in accordance
with the program quality management plan
(QMP)(4) and the test/QA plan for this
investigation. QA/quality control
procedures are summarized below.
3.1 Performance Evaluation Audit
Performance evaluation audits were
conducted to assess the quality of the results
obtained during these tests. Table 3-1
summarizes the performance evaluation
audits that were performed.
Table 3-1. Performance Evaluation Audits
Measurement
Audit
Procedure
Allowable
Tolerance
Actual
Tolerance
Volume
Temperature
Micropipettes checked by gravimetric
evaluation
Compared to independent calibrated
thermometer value
± 10%
±5°C
6 pipettes checked, all
78 temperature
comparisons, all difference
were
-------
QMP. For these tests, findings were noted
(none significant) in the data quality audit;
no follow-up corrective action was
necessary. The findings were mostly minor
data transcriptions requiring some
recalculation of efficacy results, but none
were gross errors in recording. Copies of
the assessment reports were distributed to
the EPA QA Manager and Battelle staff.
QA/quality control (QC) procedures were
performed in accordance with the test/QA
plan.
3.5 Data Review
Records generated in the investigation
received a QC/technical review and a QA
review before they were used to calculate,
evaluate, or report investigation results. All
data were recorded by Battelle staff. The
person performing the review was involved
in the tests and added his/her initials and the
date to a hard copy of the record being
reviewed. This hard copy was returned to
the Battelle staff member, who stored the
record.
3.6 Performance Criteria
As shown in Table 3-2, all inoculation
amounts were within the target range of
1 x 108 - 1 x 1010 CFU/mL (1 x 107 - 1 x
109 CPU/coupon). The percentage
recoveries of viable and culturable bacteria
from positive controls were within the target
range for all materials except for one set of
wood samples (Section 3.7).
No CPU were recovered from any blank
coupons and no results were excluded as
outliers.
3.7 Deviation
Although the recovery of B. suis from wood
during the method demonstration tests was
within the performance criteria (see Table 4-
1), the recovery from wood positive controls
was less than 5% for one set of tests
(associated with the Day 0 decontamination
results); this result is more than likely
attributed to the difficulty in recovering
organisms from porous materials during the
extraction process. Nevertheless, an
appreciable amount of B. suis was recovered
(>1 x 107 CPU/coupon) from wood to assess
persistence overtime.
Table 3-2. Persistence Testing Inoculation and Positive Control Recovery Data*
Material
Inoculation Control,
CFU/Coupon
Mean Recovered
CFU/coupon from
Positive Controls
Recovery as Percentage of
Inoculum
Aluminum
Glass
Wood
8.23xl08t
5.37xl08*
8.23 xlO8
5.37 xlO8
8.23 xlO8
5.37 xlO8
4.35 ± 0.86 xlO8
3.50±0.42xl08
4.01 ± 0.91 xlO8
2.72 ± 0.53 xlO8
4.82±4.35xl07
1.49±1.18xl07
53%
65%
49%
51%
6%
3%
*Bold value is outside the performance criteria ranges of mean CPU >5% of inoculation levels recovered from the positive
control coupons.
f Inoculation control for all persistence test samples and Day 28 decontamination test samples.
J Inoculation control for Day 0 decontamination samples.
-------
4.0 Persistence Test Results
For this investigation, persistence data were
generated for B. suis in contact with three
different materials exposed to two
environmental conditions for controlled
exposure durations. Persistence curves were
also generated, where applicable, by
graphing the B. suis log survivorship of each
material against time in days for each set of
environmental conditions. The following
sections summarize the results of the method
demonstration (tests conducted initially to
ensure sufficient B. suis recovery from each
inoculated material) and the persistence
investigation.
4.1 Method Demonstration - B. suis
Recovery
A brief method demonstration was
performed to ensure that methods previously
used for extracting biological agents from
materials were applicable for the
combinations of B. suis and material.
Results of the method demonstration are
presented in Table 4-1. The B. suis
recoveries, enumerated as CPU/coupon,
attained the recovery performance criterion
(mean CPU/coupon >5% of the inoculated
level) specified in the test/QA plan.
Recoveries of >5% of the bacteria inoculated
onto the coupon (i.e., >5.0 x 106 CPU) allow
for a sufficient amount of initial bacteria to
assess persistence. This method
demonstration was conducted at ambient
temperature and relative humidity (-22 °C
and 40% RH). No attempt was made to
control these parameters.
Table 4-1. Recovery Data from Method Demonstration
Material
Aluminum
Glass
Wood
B. suis Inoculation
Control,
CFU/coupon
4.50xl07
4.50 xlO7
7.77 xlO8
Recovered B.
CFU/coupon*
3.15±0.133xl07
2.93 ± 0.085 xlO7
6.64 ± 9.07 xlO7
suis
%*
70.1 ±2. 95
65.2 ±1.89
8.55 ±11.7
*Data are expressed as mean ± standard deviation of five replicate coupons.
4.2 Persistence Results
Persistence results for each
material/environmental condition
combination are summarized in Tables 4-2
through 4-4 and Figures 4-1 and 4-2. Where
inoculation controls are identical, the
coupons were inoculated with the same
stock suspension on the same day. An
"ND" for mean recovered logs in the result
tables indicates that no CPU were detected
in the undiluted extract sample plated for
any of five replicate coupons. A "0" was
used in the calculations of mean recovered
CFU/coupon for any replicate coupon
having no CPU detected. A single viable
bacterium present in the plated extract
would be expected to be observed as a CPU.
4.2.1 Aluminum
The results obtained for B. suis persistence
on aluminum are summarized in Table 4-2
and Figures 4-1 and 4-2. B. suis persisted
for at least 56 days (the longest duration
tested) at both environmental conditions; the
associated bacteria recoveries were higher at
low temperature (e.g., 6.35 logs at 56 days)
than at moderate temperature (e.g., 2.51 logs
at 56 days).
10
-------
Table 4-2. B. suis Persistence on Aluminum
Duration, Inoculation Control,
Days CFU/coupon
14
9Q
8.23 x 108
42
56
14
8.23 x 108
42
56
Mean Recovered B. suis,
Positive Control*
22°C±2°C;40%RH±
8.63 ±0.08
5°C±3°C;30%RH±
8.63 ±0.08
Log Survivorshipt
Test Coupon5
15%
3. 99 ±0.28
3. 45 ±0.42
2.68 ±0.37
2. 51 ±1.22
15%
7.12 ±0.19
6.70 ±0.10
6.48 ± 0.24
6.35±0.18
Mean Log Reduction*
4.64 ± 0.26
5.18±0.37
5. 95 ±0.33
6. 12 ±1.07
1.51±0.18
1.94 ±0.12
2.15 ±0.22
2.28 ±0.18
Data are expressed as mean log survivorship ± standard deviation.
*Data are expressed as mean log reduction ± 95% CI.
* Positive control coupons were inoculated and extracted at time zero (i.e., two hours 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.
4.2.2 Glass duration tested) at both environmental
conditions; the mean bacteria recoveries
The results obtained for persistence of B. were higher at low temperature (e.g., 6.25
suis on glass are summarized in Table 4-3 logs at 56 days) than at moderate
and Figures 4-1 and 4-2. On glass, B. suis temperature (e.g., 2.78 logs at 56 days).
persisted for at least 56 days (the longest
Table 4-3. B. suis Persistence on Glass
Duration, Inoculation Control, —
Days CFU/coupon
14
8.23 x 108
42
56
14
8.23 x 108
42
56
Mean Recovered B. suis,
Positive Control*
22°C±2°C;40%RH±
8.60 ±0.09
5°C±3°C;30%RH±
8.60 ±0.09
Log Survivorshipt
Test Coupon§
15%
3.81 ±0.47
3.20 ±0.19
3.06 ±0.12
2.78 ±1.09
15%
7.07 ± 0.03
6.64 ± 0.07
6.74 ± 0.27
6.25 ±0.41
Mean Log Reduction*
4.78 ± 0.42
5.40 ±0.19
5. 54 ±0.14
5. 82 ±0.96
1.52 ±0.09
1.95±0.10
1.85 ±0.25
2. 34 ±0.37
Data are expressed as mean log survivorship ± standard deviation.
*Data are expressed as mean log reduction ± 95% CI.
Positive control coupons were inoculated and extracted at time zero (i.e., two hours 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.
11
-------
4.2.3 Wood
The results obtained for persistence of B.
suis on wood are summarized in Table 4-4
and Figures 4-1 and 4-2. B. suis persisted
for at least 42 days at the moderate
temperature environmental condition (log
reduction of 7.28) and at least 56 days (the
Table 4-4. B. suis Persistence on Wood
longest duration tested; log reduction of
2.02) at the low temperature environmental
conditions. The mean bacteria recoveries
were higher at low temperature (e.g., 5.68
logs at 42 days) than at moderate
temperature (e.g., 0.40 logs at 42 days).
Duration, Inoculation Control,
Days CFU/coupon
14
28
8.23 x 108
42
56
14
28
8.23 x 108
42
56
Mean Recovered B. suis,
Positive Control*
22°C±2°C;40%RH±
7. 59 ±0.29
5°C±3°C;30%RH±
7. 59 ±0.29
Log Survivorshipt
Test Coupon§
15%
4. 18 ±0.48
0.53 ±1.19
0.40 ±0.89
ND
15%
6. 14 ±0.09
5.77 ±0.61
5.68 ±0.45
5.67 ±0.50
Mean Log Reduction-
3.40 ±0.49
7.05 ±1.08
7.19 ±0.82
>7.59±0.25
1.45 ±0.26
1.81 ±0.60
1.90 ±0.47
1.92 ±0.51
T Data are expressed as mean log survivorship ± standard deviation.
*Data are expressed as mean log reduction ± 95% CI.
* Positive control coupons were inoculated and extracted at time zero (i.e., two hours 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
.
3
I/)
Duration (Days)
-Aluminum —*—Glass
Wood
Figure 4-1 B. suis persistence at 22 °C ± 2 °C and 40% RH ± 15%.
Data are presented as mean log survivorship.
12
-------
V
14
28
Duration (Days)
-Aluminum
-Glass
42
Wood
56
Figure 4-2 B. suis persistence at 5 °C ± 3 °C and 30% RH ± 15%.
Data are presented as mean log survivorship.
13
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5.0 Decontamination Test Results
The decontamination efficacy of pH-
amended Ultra Clorox® bleach, 70% Ethanol
(EtOH), and 1% citric acid was evaluated
for B. suis on three building material
surfaces (aluminum, glass, and wood),
subjected to two environmental conditions
(22 °C ± 2 °C and 40% RH ± 15%; 5 °C ± 3
°C and 30% RH ± 15%), and two time
points (0 and 28 days). Along with the
persistence test coupons, one set of
decontamination test coupons was allowed
to incubate at the required environmental
condition for 28 days. At that time, both the
decontamination and persistence test
coupons were removed from the chamber
and processed as previously described
Section 2.6. During this investigation, no B.
suis was recovered from any of the
associated blanks. The following sections
summarize the results from the
decontamination tests.
5.1 pH-amended Ultra Clorox® Bleach
5.7.7 Neutralization Methodology
The neutralization tests indicated that the
optimal amount of STS was 1.5% for glass
and wood and 1.0% for aluminum. These
concentrations of STS yielded a percent of
control recovery of 89.8% for glass and
wood and 88.6% for aluminum. These
results are presented in Tables 5-1, 5-2, and
5-3.
Table 5-1. Spray-and-Weigh for pH-amended Ultra Clorox Bleach Deposition on Three
Outdoor Materials
Material Type
Aluminum
Glass
Wood*
Pre-Spray Avg., g
18.45
21.06
13.67
Post-Spray Avg., g
18.88
21.32
13.93
Difference Avg., g
0.43
0.26
0.26
Wood required a single re-application of the pH-amended Ultra Clorox® bleach.
Table 5-2. Neutralization Testing for pH-amended Ultra Clorox® Bleach with B. suis
Against Glass and Wood Test Materials
Treatment
pH-amended Ultra Clorox® bleach + Bacteria*
PBS + Bacteria (Control)*
pH-amended Ultra Clorox bleach + PBS + 0.5% STS + Bacteria*
pH-amended Ultra Clorox bleach + PBS + 1 .0% STS + Bacteria*
pH-amended Ultra Clorox bleach + PBS + 1 .5% STS + Bacteria*
Inoculum,
CFU
5.53xl08
5.53xl08
5.53xl08
5.53xl08
5.53xl08
Total
Observed
CFU
0
5.25 x 108
4.64 x 108
3.86xl08
4.71 xlO8
%of
Control
0
-
88.5
73.7
89.8
Total volume is 10 mL pH-amended Ultra Clorox bleach.
*Total volume is 10 mL PBS.
*Total volume is 10 mL extraction buffer + 0.26 mL pH-amended Ultra Clorox® bleach determined by the "spray-and-weigh"
"-" Not Applicable.
14
-------
Table 5-3. Neutralization Testing for pH-amended Ultra Clorox® Bleach with B. suis
Against Aluminum Test Materials
Treatment
pH-amended Ultra Clorox® bleach + Bacteria*
PBS + Bacteria (Control)*
pH-amended Ultra Clorox® bleach + PBS + 0.5% STS + Bacteria*
pH-amended Ultra Clorox® bleach + PBS + 1 .0% STS + Bacteria*
pH-amended Ultra Clorox® bleach + PBS + 1 .5% STS + Bacteria*
Inoculum,
CFU
5.53xl08
5.53xl08
5.53xl08
5.53xl08
5.53xl08
Total
Observed
CFU
0
5.25 x 108
4.08 xlO8
4.65 x 108
4.09 xlO8
%of
Control
0
-
77.8
88.6
78.0
T Total volume is 10 mL pH-amended Ultra Clorox bleach.
*Total volume is 10 mL PBS.
* Total volume is 10 mL extraction buffer + 0.43 mL pH-amended Ultra Clorox® bleach determined by the "spray-and-weigh"
"-" Not Applicable.
5.7.2 pH-Amended Ultra Clorox9 Bleach
Test Results
On each day of decontamination testing
(Day 0 and Day 28), pH-amended Ultra
Clorox® bleach was freshly prepared by
mixing 9.4 parts sterile water (Sigma
Aldrich, St. Louis, MO), 1 part Ultra
Clorox® (44600, Clorox® Company,
Oakland, CA), and 1 part 5% acetic acid (JT
Baker, Phillipsburg, NJ). A new unopened
bottle of Ultra Clorox® was used for each
day of testing. The resulting solution will
have a mean pH of-6.8 and a mean total
chlorine content of -6200 parts per million
(ppm). The average temperature of the
decontaminant at the time of testing was
22.4 °C.
At 22 °C ± 2 °C and 40% RH ± 15%, the
decontamination efficacy of pH-amended
Ultra Clorox® bleach was highest for
aluminum at both Day 0 and Day 28 (log
reductions of 8.54 and 3.45, respectively),
followed by glass (8.43 and 3.20), and wood
(1.22 and 0.53). The porous wood material
exhibited lower log reduction at the Day 0
time point, but still showed complete
inactivation of B. suis after Day 28. The
results were similar for the 5 °C ± 3 °C and
30% RH ± 15%, except that the log
reduction due to decontamination for wood
at Day 28 was 0.51 and complete
inactivation was not achieved at this
condition. The highest efficacy was
achieved on the nonporous aluminum and
glass for both environmental conditions.
These results are presented in Table 5-4 and
Figure 5-1.
15
-------
Table 5-4. Inactivation of B. suis using pH-amended Ultra Clorox® Bleach
Test
Material
Day
Inoculation
Control
(CFU/coupon)
Mean Recovered B. suis (CFU/coupon)* Mean Log Reduction*
Positive
Control*
Persistence Test Decontamination Reduction due
Coupon* Test Coupon5 to Attenuation
Reduction due
to Decont
Combined
Reduction"1"
22°C±2°C;40±15%RH
Aluminum
Glass
Wood
Aluminum
Glass
Wood
0
28
0
28
0
28
0
28
0
28
0
28
5.37 xlO8
8.23 x 108
5.37xl08
8.23 xlO8
5.37 xlO8
8.23 xlO8
5.37xl08
8.23 xlO8
5.37xl08
8.23 xlO8
5.37xl08
8.23 xlO8
3.50 ± 0.42 xlO8
4.35±0.86xl08
2.72 ± 0.53 xlO8
4.01 ± 0.91 xlO8
1.49±1.18xl07
4.82±4.35xl07
3.50±0.42xl08
4.35 ± 0.86 xlO8
2.72±0.53xl08
4.01 ± 0.91 xlO8
1.49±1.18xl07
4.82 ± 4.35 xlO7
4.06 ± 3.92 x 103
1.71±0.70xl03
9. 34 ±20. 9x10'
5°C±3°C;30±
5.07±1.27xl06
4.45 ± 0.70 xlO6
9.80 ± 7.99 xlO5
O.OOE+00
O.OOE+00 5. 18 ±0.37
O.OOE+00
O.OOE+00 5.40 ±0.19
8.26 ± 4.89 xlO5
O.OOE+00 7.05 ±1.08
15% RH
O.OOE+00
O.OOE+00 1.94 ±0.12
O.OOE+00
O.OOE+00 1.95 ±0.10
8.26 ± 4.89 xlO5
3.19±2.05xl05 1.81 ±0.60
>8. 54 ±0.05
>3.45±0.36
>8.43±0.09
>3 .20 ±0.17
1.22 ±0.34
>0. 53 ±1.05
>8. 54 ±0.05
>6.70 ± 0.09
>8.43±0.09
>6.64 ± 0.06
1.22 ±0.34
0.51 ±0.83
>8.63 ±0.07
>8.60±0.08
>7.59 ±0.25
>8.63 ±0.07
>8.60±0.08
2.33 ±0.68
* Data are expressed as mean logs recovered ± standard deviation of five replicates.
* Data are expressed as mean log reduction ± 95% CI.
* Positive control coupons were inoculated and sprayed with SFW, but not exposed to the decontaminant.
f Persistence test coupons were inoculated and exposed to the environmental condition for the exposure duration.
§ Decontamination test coupons were inoculated and exposed to the decontaminant for the contact time (15 minutes for aluminum and
glass; 30 minutes with 2 applications for wood).
f Log reduction calculated using Day 28 persistence test samples instead of positive control values (refer to Section 2.6).
+ Combined log reduction due to attenuation and decontamination of B. suis after 28 days.
> Indicates that no viable agent was recovered from test coupons. Values preceded by ">" should be considered equal, as reductions were determined by inoculum titer
and efficiency of recovery.
16
-------
Alum.
DayO
Alum. Alum.
Day 28 Day 28
Moderate Low Temp.
Temp.
Glass
Day 0
l Reduction due to Attenuation
Glass Glass Wood
Day 28 Day 28 Day 0
Moderate Low Temp
Temp.
Reduction due to Decontamination
Wood Wood
Day' 28 Day 28
Moderate LowTemp.
Temp.
Figure 5-1 Inactivation of B. suis using pH-amended Ultra Clorox® bleach.
5.2 70%Ethanol
5.2.1 Neutralization Methodology
Neutralization of the 70% EtOH was
achieved using 100% Dey Engley (D/E)
Neutralizing Broth (BD Diagnostic Systems,
Franklin Lakes, NJ).
Quantification of 70% EtOH application
was assessed using the same procedure as
described in Section 5.1.1. The 100% D/E
Neutralizing Broth yielded a control
recovery percentage of 103.7% for glass,
98.5% for aluminum, and 105.6% for wood.
These results are presented in Tables 5-5,
and 5-6.
Table 5-5. Spray-and-Weigh for 70% Ethanol Deposition on Three Outdoor Materials
Material Type
Aluminum
Glass
Wood*
Pre-Spray Avg., g
18.51
21.35
14.16
Post-Spray Avg., g
18.66
21.40
14.34
Difference Avg., g
0.15
0.05
0.18
*Wood required a single re-application of the 70% EtOH.
Table 5-6. Neutralization Testing for 70% Ethanol with B. suis Against Aluminum, Glass,
and Wood Test Materials
Treatment
70% EtOH + Bacteria1
D/E Broth + Bacteria (Control)*
0. 15 mL 70% EtOH + D/E Broth + Bacteria*
0.05 mL 70% EtOH + D/E Broth + Bacteria*
0. 18 mL 70% EtOH + D/E Broth + Bacteria*
Inoculum,
CFU
4.70 xlO8
4.70 x 108
4.70 x 108
4.70 x 108
4.70 x 108
Total
Observed
CFU
0
4.16 xlO8
4.09 xlO8
4.31xl08
4.39xl08
%of
Control
0
98.49
103.72
105.56
r Total volume is 10 mL 70% EtOH.
*Lotal volume is 10 mL D/E Broth.
Total volume is 10 mL D/E Broth+ volume 70% EtOH determined by the "spray-and-weigh"
"-" Not Applicable.
method.
17
-------
5.2.2 70% Ethanol Test Results
On each day of decontamination testing
(Day 0 and Day 28), an unopened bottle of
70% EtOH (SaniHol®ST, Decon
Laboratories, King of Prussia, PA) was
used. The average temperature of the
decontaminant at the time of testing was
21.6 °C.
At 22 °C ± 2 °C and 40 ± 15% RH, the
decontamination efficacy of 70% EtOH was
highest for aluminum at both Day 0 and Day
28 (log reductions of 8.54 and 3.45,
respectively), then glass (8.43 and 3.20), and
wood (2.15 and 0.13). At 5 °C ± 3 °C and
30 ± 15% RH, the decontamination efficacy
of 70% EtOH was also highest for
aluminum at both Day 0 and Day 28 (log
reductions of 8.54 and 6.70 respectively),
then glass (8.43 and 6.64), and wood (2.15
and 3.26). Complete inactivation was
achieved on aluminum and glass at both
environmental conditions. The highest
efficacy was achieved on the nonporous
materials, aluminum and glass. Interestingly,
at low temperature EtOH was more effective
at inactivating B. suis than pH-adjusted
bleach. These results are presented in Table
5-7 and Figure 5-2.
18
-------
Table 5-7. Inactivation of B. suis using 70% Ethanol
Test
Material
Day
Inoculation
Control
(CFU/coupon)
Mean Recovered B. suis (CFU/coupon)* Mean Log Reduction*
Positive
Control*
Persistence Test
Coupon*
Decontamination Reduction due
Test Coupon5 to Attenuation
Reduction due
to Decont
Combined
Reduction"1"
22°C±2C;40±15%RH
Aluminum
Glass
Wood
Aluminum
Glass
Wood
0
28
0
28
0
28
0
28
0
28
0
28
5.37 xlO8
8.23 x 108
5.37xl08
8.23 x 108
5.37xl08
8.23 x 108
5.37xl08
8.23 x 108
5.37xl08
8.23 x 108
5.37xl08
8.23 x 108
3.50 ± 0.42 xlO8
4.35±0.86xl08
2.72 ± 0.53 xlO8
4.01 ± 0.91 xlO8
1.49±1.18xl07
4.82±4.35xl07
3.50±0.42xl08
4.35 ± 0.86 xlO8
2.72±0.53xl08
4.01 ± 0.91 xlO8
1.49±1.18xl07
4.82 ± 4.35 xlO7
4. 06 ±3. 92
1.71 ±0.70
9. 34 ±20. 9
5°C±3
5.07 ±1.27
4.45 ±0.70
9.80 ±7.99
xlO3
xlO3
xlO1
O.OOE+00
O.OOE+00 5. 18 ±0.37
O.OOE+00
O.OOE+00 5.40 ±0.19
4.46±6.12xl05
2. 00 ±4.47x10' 7.05 ±1.08
>8. 54 ±0.05
>3.45±0.36
>8.43±0.09
>3.20±0.17
2.15 ±0.95
0.13±1.31
>8.63 ±0.07
>8.60±0.08
7.19 ±0.82
°C;30±15%RH
xlO6
xlO6
xlO5
O.OOE+00
O.OOE+00 1.94 ±0.12
O.OOE+00
O.OOE+00 1.95 ±0.10
4.46±6.12xl05
2.29±3.18xl04 1.81 ±0.60
>8. 54 ±0.05
>6.70 ± 0.09
>8.43±0.09
>6.64 ± 0.06
2.15 ±0.95
3.26 ±2.17
>8.63 ±0.07
>8.60±0.08
5.07±2.11
* Data are expressed as mean logs recovered ± standard deviation of five replicates.
* Data are expressed as mean log reduction ± 95% CI.
* Positive control coupons were inoculated and sprayed with SFW, but not exposed to the decontaminant.
f Persistence test coupons were inoculated and exposed to the environmental condition for the exposure duration.
§ Decontamination test coupons were inoculated and exposed to the decontaminant for the contact time (15 minutes for aluminum and
glass; 30 minutes with 2 applications for wood).
f Log reduction calculated using Day 28 persistence test samples instead of positive control values (refer to section 2.6).
+ Combined log reduction due to attenuation and decontamination of B. suis after 28 days.
> Indicates that no viable agent was recovered from test coupons. Values preceded by ">" should be considered equal, as reductions were determined by inoculum titer
and efficiency of recovery.
19
-------
Glass
Day 28
Moderate
Temp
Glass
Day 28
Low Temp.
Wood
DayO
Wood
Day 28
Moderate
Temp
Wood
Day 28
LowTemp.
I Reduction dueto Attenuation Reduction dueto Decontamination
Figure 5-2 Inactivation of B. suis using 70% ethanol.
5.3. 1% Citric Acid
5.3.1 Neutralization Methodology
Neutralization of the 1% citric acid was
achieved with 100% D/E Neutralizing Broth
(BD Diagnostic Systems, Franklin Lakes,
NJ).
The amount of 1% citric acid applied to the
coupons was assessed using the same
procedure previously described in Section
5.1.1. The 100% D/E Neutralizing Broth
yielded a control recovery percentage of
102.3% for aluminum, 96.7% for glass, and
102.2% for wood and was determined to be
effective in neutralizing 1% citric acid.
These results are presented in Tables 5-8
and 5-9.
Table 5-8. Spray- and-Weigh for 1% Citric
Material Type
Aluminum
Glass
Wood*
Pre-Spray Avg., g
18.71
21.35
14.16
Acid Deposition on Three Outdoor Materials
Post-Spray Avg., g
19.28
21.63
14.41
Difference Avg., g
0.57
0.28
0.25
Wood required a single re-application of 1% citric acid.
20
-------
Table 5-9. Neutralization Testing for 1% Citric Acid with B. suis Against Aluminum,
Glass, and Wood Test Materials
Treatment
1% Citric Acid + Bacteria1
D/E Broth + Bacteria (Control)*
0.57 mL 1% Citric Acid + D/E Broth + Bacteria*
0.28 mL 1% Citric Acid + D/E Broth + Bacteria*
0.25 mL 1% Citric Acid + D/E Broth + Bacteria*
Inoculum,
CFU
4.57xl08
4.57xl08
4.57xl08
4.57 xlO8
4.57 xlO8
Total
Observed
CFU
0
4.12xl08
4.22 xlO8
3.99xl08
4.21 xlO8
%of
Control
0
-
102.28
96.65
102.17
T Total volume is 10 mL 1% citric acid.
*Total volume is 10 mL D/E Broth.
* Lotal volume is 10 mL D/E Broth + volume 1% citric acid determined by the "spray-and-weigh."
"-" Not Applicable.
5.3.2 1% Citric Acid Test Results
On each day of decontamination testing
(Day 0 and Day 28), 1% citric acid was
freshly prepared by adding 1 g citric acid,
anhydrous, (> 99.5% purity; Sigma Aldrich)
to 99 mL hard water until completely
dissolved. The average temperature of the
decontaminant at the time of testing was
21.9 °C.
At 22 °C ± 2 °C and 40% RH ± 15%, the
decontamination efficacy of 1% citric acid
was highest for aluminum at both Day 0 and
Day 28 (log reductions of 1.78 and 3.45,
respectively), then glass (1.30 and 3.20), and
wood (0.76 and -0.29). At 5 °C ± 3 °C and
30% ± 15% RH, log reductions due to
decontamination were also highest for
aluminum (1.78 and 5.69) and glass (1.30
and 3.75) at Days 0 and 28, respectively.
Log reductions on wood were 0.76 and 0.38
for 0 and 28 days, respectively. The highest
efficacy was observed on the nonporous
materials, aluminum and glass. Also, higher
log reductions were achieved at the
moderate temperature and relative humidity
conditions when compared to the low
temperature and relative humidity
conditions. Overall, 1% citric acid was less
effective, regardless of temperature or
material, than 70% EtOH or pH-adjusted
bleach. These results are presented in Table
5-10 and Figure 5-3.
21
-------
Table 5-10. Inactivation of B. suis using 1% Citric Acid
Test
Material
Day
Inoculation
Control
(CFU/coupon)
Mean Recovered B. suis (CFU/coupon)*
Positive
Control*
Persistence Test
Coupon*
Decontamination
Test Coupon5
Mean Log Reduction*
Reduction due
to Attenuation
Reduction due
to Decont
Combined
Reduction"1"
22±2°C;40±15%RH
Aluminum
Glass
Wood
Aluminum
Glass
Wood
0
28
0
28
0
28
0
28
0
28
0
28
5.37xl08
8.23 xlO8
5.37xl08
8.23 xlO8
5.37xl08
8.23 xlO8
5.37xl08
8.23 xlO8
5.37xl08
8.23 xlO8
5.37xl08
8.23 xlO8
3.50±0.42xl08
4.35 ± 0.86 xlO8
2.72±0.53xl08
4.01±0.91xl08
1.49±1.18xl07
4.82±4.35xl07
3.50±0.42xl08
4.35±0.86xl08
2.72 ± 0.53 xlO8
4.01 ± 0.91 xlO8
1.49±1.18xl07
4.82 ± 4.35 xlO7
4.06±3.92xl03
1.71±0.70xl03
9.34*20.9x10'
5±3°C;30±
5.07±1.27xl06
4.45 ± 0.70 xlO6
9.80 ± 7.99 xlO5
6.60 ± 4.09 xlO6
O.OOE+00
1.40±0.49xl07
O.OOE+00
2.39±1.19xl06
4.66 + 6.49x10'
15% RH
6.60 ± 4.09 xlO6
3.39±7.22xl02
1.40±0.49xl07
2.08 ± 2.08 xlO3
2.39±1.19xl06
5.11±3.43xl05
5.18±0.37
5.40 ±0.19
7.05 ±1.08
1.94 ±012
1.95±0.10
1.81 ±0.60
1.78 ±0.22
>3.45±0.36
1.30±0.16
>3.20±0.17
0.76 ±0.35
-0.29 ±1.44
1.78 ±0.22
5. 69 ±1.29
1.30±0.16
3. 75 ±0.75
0.76 ±0.35
0.38 ±0.94
>8.63 ±0.07
>8. 60 ±0.08
6.76 ±1.02
7.62 ±1.29
5.70 ±0.75
2.19 ±0.81
* Data are expressed as mean logs recovered ± standard deviation of five replicates.
* Data are expressed as mean log reduction ± 95% CI.
* Positive control coupons were inoculated and sprayed with SFW, but not exposed to the decontaminant.
f Persistence test coupons were inoculated and exposed to the environmental condition for the exposure duration.
§ Decontamination test coupons were inoculated and exposed to the decontaminant for the contact time (15 minutes for aluminum and
glass; 30 minutes with 2 applications for wood).
f Log reduction calculated using Day 28 persistence test samples instead of positive control values (refer to section 2.6).
+ Combined log reduction due to attenuation and decontamination of B. suis after 28 days.
> Indicates that no viable agent was recovered from test coupons. Values preceded by ">" should be considered equal, as reductions were determined by inoculum titer
and efficiency of recovery.
22
-------
Alum Alum. Alum. Glass
Day 0 Day 28 Day 28 Day 0
Model-ate Low Temp.
Temp.
• Reduction due to Attenuation
Glass Glass
Day 28 Day 28
Moderate Low Temp.
Temp.
Wood
Day 0
Wood Wood
Day 28 Day 28
Moderate Low Temp.
Temp.
Reduction dueto Decontamination
Figure 5-3. Inactivation of B. suis using 1% citric acid.
5.4. Surface Damage to Materials
At the onset and after completion of each
decontamination test, the procedural blanks
were visually compared to the laboratory
blanks and test coupons were visually
compared to positive controls to assess any
impact the liquid decontaminants may have
had on each material type. Based on the
visual appearance of the decontaminated
coupons, there were no apparent changes in
the color, reflectivity, or roughness of
aluminum or glass after being exposed pH-
amended Ultra Clorox® bleach, 70% EtOH
or 1% citric acid for up to 15 minutes, or
wood after being exposed for up to 30
minutes.
23
-------
6.0 Summary
When exposed to 22 °C ± 2 °C and 40% RH
± 15%, B. suis persisted on aluminum and
glass coupons for at least 56 days, the
longest duration tested (log reductions
ranged from 4.64 to 6.12). B. suis persisted
on wood for up to 42 days (log reductions
ranged from 3.40 to 7.59); however, no
viable bacteria were recovered after 56 days
under this environmental condition. When
exposed to 5 °C ± 3 °C and 30% RH ± 15%,
B. suis persisted on aluminum, glass, and
wood for up to 56 days (log reductions
ranged from 1.51 to 2.34).
On Days 0 and 28, pH-amended Ultra
Clorox® bleach completely inactivated B.
suis on aluminum (log reductions of 8.54
and 8.63, respectively) and glass (log
reductions of 8.43 and 8.60, respectively)
under both sets of environmental conditions.
At 22 °C ± 2 °C and 40% RH ± 15%, no
viable B. suis was recovered from the wood
coupons after 28 days of exposure (log
reduction of 7.59). However, bacteria were
recovered from the wood coupons on Day 0
(for both environmental conditions) and at
Day 28 for the 5 °C ± 3 °C and 30% ± 15%
RH environmental condition (log reductions
were 1.22 and 2.33, respectively).
B. suis was completely inactivated by 70%
EtOH at Days 0 and 28 on aluminum (log
reductions of 8.54 and 8.63) and glass (log
reductions of 8.43 and 8.60) under both sets
of environmental conditions. Viable B. suis
was recovered from wood on Day 0 (log
reduction of 2.15) and on Day 28 for both
environmental conditions (log reduction of
7.19 for moderate temperature and 5.07 for
low temperature).
After being decontaminated with 1% citric
acid, no viable B. suis was recovered from
aluminum (log reduction of 8.63) or glass
(log reduction of 8.60) after being held for
28 days at the moderate temperature
environmental condition. At the low
temperature environmental condition, B. suis
was recovered following application of 1%
citric acid after 28 days from aluminum,
glass, and wood (log reductions of 7.62,
5.70, and 2.19, respectively). For both
environmental conditions, bacteria were
recovered from aluminum, glass, and wood
(log reductions of 1.78, 1.30, and 0.76,
respectively) on Day 0.
Decontamination with pH-amended Ultra
Clorox® bleach, 70% EtOH, or 1% citric
acid did not cause visible damage to
aluminum or glass coupons when exposed
for up to 15 minutes or to wood when
exposed for up to 30 minutes.
These data suggest that temperature and
relative humidity may play a vital role in the
persistence of B. suis on aluminum, glass,
and wood building materials and that B. suis
will survive for extended periods of time (up
to 56 days) in the environment. They also
suggest that the effectiveness of a
decontaminant is higher on nonporous
materials compared to porous materials.
These data suggest that the material type in
which the biological agent is in contact is an
important factor for decontaminant
technology selection. Also, for difficult to
decontaminate materials, disinfectants may
be as effective as sporicides at inactivating
vegetative threat agents. For example, pH-
Amended Ultra Clorox® bleach (a sporicidal
decontaminant) was no more effective than
70% EtOH (a disinfectant) on these
materials. Overall, 1% citric acid was the
least effective decontaminant tested.
24
-------
7.0 References
1. Quality Assurance/Test Plan for
Persistence Testing ofBrucella suis on
Outdoor Materials, October, 2008, US
EPA (available upon request).
2. Sarinas, P.S.A. and R.K. Chitkara.
Brucellosis. Semin. Respir. Infect.,
2003(18): 168-182.
3. Standard Practice for Maintaining
Constant Relative Humidity by Means of
Aqueous Solutions. ASTM
International, October, 2006.
4. Quality Management Plan (QMP ) for
the National Homeland Security
Research Center. US EPA. (available
upon request).
25
-------
APPENDIX A
26
-------
FINAL CONTRIBUTING SCIENTIST REPORT
on
Confirmation OfBrucella suis 1330 Identity by Whole Genome Sequencing
In support of Battelle Study Number EPC-10-001
May 2011
by
Richard L. Warren, Ph.D.
fl(
Richard L. Warren, Ph.D. Date
Research Leader
6/1
7 It
Gabriel T. Meister/Ph.D. Date
Manager, Bacteriology & Toxinology
Battelle Biomedical Research Center
MAILING ADDRESS
Battelle Biomedical
Research Center (BBRC)
505 King Ave., JM-7
Columbus, OH 43201-2696
27
-------
EXECUTIVE SUMMARY
The identity of Battelle's working stock of B. suis 1330 (ATCC23444) was confirmed by whole
genomic sequencing. Genomic DNA was extracted from samples of the working stock and
sequencing libraries prepared using NextEra™ DNA sample kits. The genomic library was
sequenced on an Illumina platform by OSU Comprehensive Cancer Center NASR-lllumina
Core. The sequence reads were mapped to the sequences for 5. suis
1330 published in the National Centers for Bioinformatic Information (NCBI accession numbers
NC_Q04310 and NC_004311) with CLC Genomic Workbench v 4.6.1. Greater than 99% of
33,451,656 total reads with an average read length of 72 bases mapped to the reference
sequences. The average coverage for chromosome 1 (2,107,794 bp) was 738 reads per base and
for chromosome 2 (1,207,381 bp) was 639 reads per base. The consensus sequences for
chromosome 1 and 2 confirmed that Battelle's working stock was B. suis 1330.
28
-------
TABLE OF CONTENTS
Page
EXECUTIVE SUMMARY ii
ABBREVIATIONS iv
1.0 INTRODUCTION 1
2.0 METHODS 1
2.1 Strains and Culture Conditions 1
2.2 NextEra™ DNA Sample Preparation Kit 1
2.3 Illumina Sequencing 2
2.4 Bioinfbrmatics 2
3.0 RESULTS 2
4.0 SUMMARY 3
5.0 REFERENCE LIST 5
LIST OF TABLES
Table 1 Nucleotide Variance of the Consensus Sequence of the Illumina Reads Aligned
with NC_Q04310/NC_004311 as the Reference 4
Table 2 Comparision of the Consensus Sequence of the Illumina Reads with the Genomes
of Other Brucella Species 5
111
29
-------
ABBREVIATIONS
ATCC American Type Culture Collection
BHI Brain Heart Infusion
DIPs insertions or deletions
HMT NextEra™ reaction buffer
HMW high molecular weight
NCBI National Center for Biotechnology Information
PCR Polymerase Chain Reaction
SBS Sequencing by Synthesis
SNPs single nucleotide polymorphisms
TE Tris-EDTA
IV
30
-------
1.0 INTRODUCTION
Brucella species carry highly conserved genomes making phylogenetic relationships
difficult to determine using molecular assays that rely on a limited number of target sites such as
PCR, DNA finger-printing or multilocus sequencing. Foster et al, (Foster et al,, 2009)
demonstrated that whole genome sequencing could be used to establish the phylogeny of
Brucella species based upon differences in single nucleotide polymorphisms. Battelle used whole
genome sequencing to confirm mat the working stock used to support studies for the sponsor was
B. suis 1330 American Type Culture Collection (ATCC) number 23444. The identity of the B.
suis 1330 strain was confirmed by whole genome sequencing of a DNA library with an Illumina.
2.0 METHODS
2.1 Strains and Culture Conditions
B. suis 1330 ATCC 23444 was cultured on Brain Heart Infusion (BHI) agar (BD
Bioscience, 2350 Qume Drive San Jose, CA 95131 Cat #: 221570) and BHI broth (BD Cat #:
211059) at 37°C ± 2°C. The bacterial culture was incubated with shaking (200 rpm) until an
increase in turbidity was observed indicating B. suis replication (overnight). Genomic DNA was
extracted with a Gentra Puregene kit (QIAgene, 27220 Turnberry Lane, Suite 200 Valencia, CA
91355). 255 ng/mL was recovered and had an OD260/280 ratio of 1.84.
2.2 NextEra™ DNA Sample Preparation Kit
(Epicentre Biotechnologies, 726 Post Road,Madison, WI 53713)
255 ng/mL of double stranded B. suis 1330 DNA was diluted 1/10 in Tris-EDTA (TE)
buffer (lOmM Tris-HCl, pH 8.0, O.lmM EOT A, pH 8.0, sterile solution, Teknova, 2290 Bert
Dr.Hollister, CA 95023). The genomic library was prepared according to the Epicentre
NextEra™ kit instruction using the high molecular weight (HMW) buffer. Briefly 13 uL of
Nuclease-Free water (Accugene), 2 uL 1/10 diluted!?, suis 1330 DNA, 4 uL NextEra™ reaction
buffer (HMT), 1 uL NextEra™ Enzyme Mix was combined and then incubated at 55 "C for 5
minutes. The DNA products are purified with MinElute Reaction Cleanup kit according to the
manufacturer's instruction (QIAgene, 27220 Turnberry Lane, Suite 200 Valencia, CA 91355).
The DNA was eluted from the columns with TE buffer. Next NextEra™ bar-code PCR
compatible sites are added by combining 17uL Nuclease-Free water, 5 uL of the purified
31
-------
fragmented DNA, 25 uL 2X NcxlEra™ (PCR) buffer, 1 uL SOX NcxtEra™ Primer Cocktail, 1
uL SOX NextEra™ Adaptor 2, 1 uL NextEra™ PCR Enzyme and the thcrmocycling conditions
were 72 C for 3 minutes, 95 C for 30 seconds, followed by 9 cycles of 95 C for 10 seconds,
62"C for 30 seconds, 72 "C 3 minutes. The reaction products were purified using MitiElute
Reaction Cleanup kit. The resulting library was characterized with an Agilent Bioanlayzer
(Appendix 1).
2.3 Illumina Sequencing
The run was performed on Illumina GAIIx system using the sequencing by synthesis
(SBS) reagent v.5 to increase the read length. The data were acquired using RTA1.8 software.
2.4 Biointbi matics
The Illumina read file (110417_s_4__l_qseq)was uploaded into CLCbio Genomic
Workbench Software (CLC bio, 10 Rogers St # 101, Cambridge, Massachusetts 02142) and
assembled against two reference genomes (NC 004310 and NC 004311) using the default
settings. Both single micleotide polymorphisms (SNPs) and deletion insertion polymorphisms
(Oil's) modules were preformed on the assembled data.
The quality of the Illumina reads was determined with FastQC™ software
(hUjr \v\v\v,hiom^^
Nucmer a subprogram of the MUMmer 3 (SourceForge.net.project.page) was used to
compare the consensus sequences for chromosome 1 and 2 with the published gcnomic
sequences ofBrucella species National Center for Biotechnology Information (NCB1). Ilie
command line statement used to execute Nucmer was nucmer -maxmatch -c 100 -p output_filc
referencc.fasta consensus.fasta.
3.0 RESULTS
Whole genome sequencing produced 33, 451,656 reads with an average length of 72
bases (Appendix 2). "Hie Illumina qseq file was imported into CLCbio Genome Workbench and
assembled using NC_010167/NC_010169 (B. mis), NC_004310/NC_004311{5. suis) and
NC 003317/NC 00318 (B, melitensis) as reference genomes. There were two different
sequences published in the NCBI database forB. suis 1330 ATCC23445
(NC_004310/NC_004311 and NC 010167/NC 010169). The alignment of the reads to
32
-------
NC_004310 and NC_004311 produced consensus sequences for chromosome 1 and 2 with the
fewest SNPs or DIPs. The consensus sequence for this alignment is found in Appendix D. Table
1 shows that six of the base changes resulted in two amino acid changes and four frame-shifts,
three additional variants were silent and variants four mapped to intragenic regions. In contrast,
if NC_010167/NC_10169 were used as reference sequences to align the Illumina reads, resulting
in 4906 SNPs between the consensus sequence and the reference genome sequences. In addition,
there were 266 deletion or insertions differences between the reference genomes and the
consensus sequence.
Foster et al (Foster et al., 2009) compared the phylogeny of thirteen strains ofBrucella
using whole genome sequencing. B. ovis was the first linage to split from the Brucella group.
Consistent with their data, B. ovis had the most genetic variation when compared with the
consensus sequences. As expected the consensus sequences for chromosome 1 and 2 less
divergent for B. mis (NC_01067/NC_010169) andB. canis (NC_010103/NC_010104) (Table 2).
4.0 SUMMARY
Whole genome sequencing identified the B. suis 1330 strain used by Battelle was almost
identical to the published sequence in the NCBI database for NC_004310 and NC_004311. The
Battelle strain of B. suis 1330 had six variations that resulted in two amino acid changes and four
frame shifts, two were silent and four were in intragenic regions.
33
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B
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1-S
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C£
C
-_J
•§ o
P
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M
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O
^
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-------
Table 2 f'ompiirisimi of the Consensus Sequence of the I Hum in.i Reads with the Genomes
of Other Brucella species
NCI31 Genome
NC 003317
NC 003318
NC_004310
NC 004311
NC 007618
NC_007624
NC_010103
NC 010104
NC_010167
NC_010169
NC_010742
NC 010742
NCJM2441
NC_()12442
NC 013119
NC_013118
NC_009505
NC 1)09504
ATCC #
23456
23456
23444
23444
23365
23365
23445
23445
23457
23457
25840
25X40
Species
B. melitetixi.i 16M
B. melitensis 16M
B_A-«JA-_1330
B suix 1330
B ai»ort«5 2308
B abortus 230S
fi. cams
B. canis
B. suis_1330
B. suis_1330
B. abortus JAW
B. abortus SI 9
B. melilensix
H. melilensLi
B. microti CCM 4915
B. microti_CCM_49\5
B. avis
B. avis
Chrom sosora e
1
"J
1
•^
1
7
1
•^
1
2
1
•}
I
2
1
-^
1
"i
SNPs
6214
4634
11
?
5871
3702
1765
1422
487
15
5882
3697
4763
4( >< )i )
3312
2354
6544
4793
The published (renoine Sequence?; were downloaded from INCH1 as fasta files and compared to the consensus assemblies with IMiicme
5.0 REFERENCE LIST
1. Foster,J.T., Beckstrom-Sternberg,S.M., Pearson,!'., Beckstrom-Stemberg,J.S., Chain.P.S.,
Roberto.F.F., HnathJ., Brettin,T., and Keim,P. (2009). Whole-genome-based phylogeny and
divergence oi'the genus Brucella. J. Bacteriol. 191, 2864-2870.
35
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