EPA/600/R-14/074 |June 2014 | www.epa.gov/ord
United States
Environmental Protection
Agency
Persistence of Categories
A and B Select Agents in
Environmental Matrices
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-14/074
June 2014
Persistence of Categories A and B Select Agents in
Environmental Matrices
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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Disclaimer
The U.S. Environmental Protection Agency (US EPA), through its Office of Research and Development,
funded and managed this literature review in collaboration with the Defense Threat Reduction Agency
and the Department of Homeland Security under the Battelle/Chemical, Biological, Radiological, and
Nuclear Defense Information and Analysis Center Contract No. SP0700-00-D-3180, Technical Area Task
CB-11-0232. It has been subjected to the Agency's peer and administrative review and has been
approved for publication as an EPA document. Note that approval does not signify that the contents
necessarily reflect the views and opinions of the EPA. Reference herein to any specific commercial
product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily
constitute or imply its endorsement, recommendation, sale, or favoring by the EPA.
This report was generated using references (secondary data) that could not be evaluated for accuracy,
precision, representativeness, completeness, or comparability and therefore no assurance can be made
that the data extracted from these publications meet US EPA's stringent quality assurance
requirements.
Questions concerning this document or its application should be addressed to:
Worth Calfee
U.S. Environmental Protection Agency
National Homeland Security Research Center
109 TW Alexander Dr
Research Triangle Park, NC 27711
919-541-7600
Calfee.Worth@epa.gov
Erin Silvestri
U.S. Environmental Protection Agency
National Homeland Security Research Center
26 W. Martin Luther King Drive, MS NG16
Cincinnati, OH 45268
513-569-7619
Silvestri.Erin@EPA.gov
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Contents
Disclaimer i
List of Acronyms and Abbreviations v
Acknowledgments vi
Executive Summary vii
1. Introduction 1
2. Purpose 1
3. Methods 2
4. Bacillus anthracis Vegetative Cell Persistence 4
4.1. B. anthracis Vegetative Cells on Fomites 5
4.2. B. anthracis Vegetative Cells in Soil 7
4.3. B. anthracis Vegetative Cells in Water 8
4.4. B. anthracis Vegetative Cell Persistence Gaps 9
5. Brucella species Persistence 9
5.1. Brucella species on Fomites 10
5.2. Brucella species in Soil 12
5.3. Brucella species in Water 13
5.4. Brucella species Persistence Gaps 14
6. Burkholderia mallei Persistence 15
6.1. B. mallei on Fomites 15
6.2. B. mallei in Water 15
6.3. B. mallei Persistence Gaps 16
7. Burkholderia pseudomallei Persistence 17
7.1. B. pseudomallei on Fomites 17
7.2. B. pseudomallei in Soil 18
7.3. B. pseudomallei in Water 21
7.4. B. pseudomallei Persistence Gaps 24
8. Coxiella burnetii Persistence 25
8.1. C. burnetii in Aerosols 25
8.2. C. burnetii on Fomites 26
8.3. C. burnetii in Soil 26
8.4. C. burnetii Persistence Gaps 27
9. Francisella tularensis Persistence 27
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9.1. F. tularensis in Aerosols 28
9.2. F. tularensis on Fomites 29
9.3. F. tularensis in Water 30
9.4. F. tularensis Persistence Gaps 35
10. Viral Encephalitis and Hemorrhagic Fever Agents Persistence 35
10.1. Viral Encephalitis and Hemorrhagic Fever Agents in Aerosols 35
10.2. Viral Encephalitis and Hemorrhagic Fever Agents on Fomites 37
10.3. Viral Encephalitis and Hemorrhagic Fever Agents in Water 41
10.4. Viral Encephalitis and Hemorrhagic Fever Agents Persistence Gaps 43
11. Yersinia pestis Persistence 43
11.1. Y. pestis in Aerosols 43
11.2. Y. pestis on Fomites 44
11.3. X. pestis in Soil 44
11.4. Y. pestis in Water 45
11.5. Y. pestis Persistence Gaps 48
12. Summary 48
13. References 55
List of Tables
Table ES-1. Agents Investigated and Number of Persistence Studies Identified vii
Table 1. Agents Investigated 2
Table 2. Document Evaluation Categories and Data Quality Indicators 4
Table 3. Bacillus Species (Vegetative Cells) Persistence on Fomites 7
Table 4. B. suis (ATCC 23444) Persistence on Fomites 11
Table 5. Brucella Species Persistence in Water 144
Table 6. B. mallei Persistence in Water 16
Table 7. B. pseudomallei Persistence on Fomites from Shams et al. (2007) 188
Table 8. B. pseudomallei Persistence in Soil 19
Table 9. B. pseudomallei Persistence in Water 224
Table 10. F. tularensis LVS Persistence on Fomites 302
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Table 11. F. tularensis Persistence in Water 335
Table 12. Viral Encephalitis and Hemorrhagic Fever Agents Persistence in Aerosols 38
Table 13. Viral Encephalitis and Hemorrhagic Fever Agents Persistence on Fomites 40
Table 14. Viral Encephalitis and Hemorrhagic Fever Agents Persistence in Water 424
Table 15. Y. pestis Persistence in Soil 47
Table 16. /. pestis Persistence in Water 49
Table 17. Summary of Agent Persistence inthe Environment 502
IV
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List of Acronyms and Abbreviations
ATCC® American Type Culture Collection (ATCC) [Manassas, VA]
BHI brain heart infusion
CDC Centers for Disease Control and Prevention
CPU colony forming units
CHAB cystine heart agar blood
gfp green fluorescent protein
HEPA high-efficiency particulate air
LCV large-cell variants
LVS live vaccine strain
mS millisiemens
PBS phosphate buffered saline
PCR polymerase chain reaction
PFU plaque forming units
ppm parts per million
RH relative humidity
SCV small-cell variants
SDC small dense cells
SERRA Support for Environmental Rapid Risk Assessment
T4 time needed to decrease the viral load by 4 logic
T99 time required for the initial titer to decrease by 99%
u.L microliter
US EPA U.S. Environmental Protection Agency
UV ultraviolet
uW microwatt
VEE Venezuelan equine encephalitis (or encephalomyelitis)
VBNC viable but nonculturable
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Acknowledgments
The following individuals and organizations are acknowledged for their contributions to this report:
U.S. Environmental Protection Agency, Office of Research and Development, National Homeland
Security Research Center
Charlena Bowling
Eletha Brady-Roberts
Worth Calfee
Wendy Davis-Hoover
Kathy Hall
Marissa Mullins
Erin Silvestri
Joseph Wood
Battelle, Contractor for the U.S. Environmental Protection Agency
VI
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Executive Summary
Persistence is the ability of an organism to remain viable (e.g., to remain alive or in the case of viruses to
remain infective) overtime under a given environmental condition and medium. Understanding a
bioterrorism agent's ability to persist within the environment will help properly assess and respond to
the agent's release. The purpose of this study was to summarize persistence data generated since the
*i
literature review conducted by Sinclair et al. (2008) as well as review the literature for persistence data
on agents not addressed by Sinclair et al. (2008). These data are critically important for at least two
reasons; 1) to allow informed emergency response and remediation decisions following a contamination
incident, and 2) to identify gaps in the current state of the science, and to focus research toward closing
these identified gaps. The scope of this literature review generally focused on agents that were
determined to be viable by culturing on artificial media and/or infecting tissue cultures. In some cases
evidence of an agent entering a viable but nonculturable state was discussed. Relevant English language
literature was identified primarily by searching each agents name and the terms "persistence",
"recoverable", "survival", "survivability", and "viability" using Google Scholar. Table ES-1 identifies the
agents covered in this review, identifies the number of studies reviewed by agent if addressed by
Sinclair et al. (2008), and lists the number of studies (excluding Sinclair et al. [2008] and works cited
therein) presenting original quantitative persistence data that were summarized in this report by
environmental media (i.e., aerosol, fomite, soil, and water). A total of 94 sources were reviewed for this
report including ecologically-based studies and literature reviews (not shown on Table ES-1) and
excluding Sinclair et al. (2008) and works cited therein.
Table ES-1. Agents Investigated and Number of Persistence Studies Identified
Agent Reviewed
Number of Studies
Included in the
Review by
Sinclair et al. (2008)"
Number of Studies Included in this Reviewt
Aerosol Fomite Soil Water
Bacillus anthracis
(vegetative cells only)
Brucella species (e.g., suis,
melitensis, abortus, etc.)
Burkholderia mallei
Burkholderia pseudomallei
Coxiella burnetii
Francisella tularensis
Viral encephalitis and
hemorrhagic fever agents
Yersinia pestis
2
Not reviewed
Not reviewed
Not reviewed
Not reviewed
6
6
0
0
0
0
1
0
3
0
1
0
2
1
o
0
5
1
0
3
o
3
7
0
6
4
Source: Sinclair, 2008, Applied and Environmental Microbiology 74(3):555-563
* The number of studies cited by Sinclair et al. (2008) presenting quantitative persistence data.
t The number of studies (excluding Sinclair et al. [2008] and works cited therein) presenting original quantitative persistence
data; ecologically-based studies and literature review data are included in this review but not reflected in this table.
1 Sinclair, R. et al., 2008. Applied and Environmental Microbiology 74(3):555-563.
VII
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The summary that follows provides a quick indication of the durations that the agents could persist in
the four types of environmental media, although "fomite" (i.e., surfaces or materials likely to carry
infection) represents many different kinds of materials/surfaces. Some data documented survival of a
given duration, but did not document survival until extinction. So, actual persistence could exceed the
persistence documented in the literature. Data for survival of the bacterial agents listed in Table ES-1
for an aerosol medium were scant; F. tularensis survived 5 days and Y. pestis survived 57 minutes. The
data for bacterial agent survival on fomites were more complete, including B. anthrads (vegetative cells
only), Brucella species, Burkholderia mallei, Burkholderia pseudomallei, and Y. pestis. Literature values
ranged from 3 days (6. pseudomallei) to 3 months (6. mallei). For survival in soil, data were available for
Brucella species, B. pseudomallei, C. burnetii, and Y. pestis. Literature values ranged from 20 days (C.
burnetii) to 30 months (6. pseudomallei). For survival in water, we found survival data for bacterial
agents B. anthracis, Brucella species, B. mallei, B. pseudomallei, F. tularensis, and Y. pestis. Literature
values ranged from 6 days (6. anthracis, vegetative cells only) to 16 years (6. pseudomallei). For viral
encephalitis and hemorrhagic fever agents, persistence was documented for aerosol medium at 120
days, for fomites at 5 days, and for water at 69 days.
Persistence durations are likely affected by the agent species and strain evaluated, the agent
preparation/application methods, media specific differences (e.g., water/soil chemistry, porous or
nonporous fomite surface types, etc.), and the methods used to recover and quantify the agent. The
environmental conditions (e.g., temperature, relative humidity) during the persistence testing are also
major drivers with regard to agent survivability. Sunlight and elevated temperatures were often
reported to shorten the persistence duration of several agents including B. mallei, Brucella species, and
viral encephalitis and hemorrhagic fever agents. Numerous studies indicate that other agents (e.g., 6.
pseudomallei and Y. pestis) seem to prefer warmer conditions. Moist conditions were found to improve
B. abortus, B. mallei, and B. pseudomallei persistence compared to more dry environments.
Following the release of an agent, other organisms/bacteria may adversely affect the persistence of the
agent or enhance the agent's survival in the environment. For example, in recent studies the soil
bacterium, Burkholderia multivorans, appeared to inhibit B. pseudomallei growth, and the bacterium,
Hylemonella gracilis, eventually dominated a filtered river water sample, apparently by inhibiting the
long-term survival of Y. pestis. However, other recent studies have shown that amoebae (e.g.,
Acanthamoeba castellanii) may provide a niche for the environmental survival of B. anthracis, B.
pseudomallei, C. burnetii, and F. tularensis. A 2006 study indicated that the rhizosphere may enhance
the proliferation of vegetative B. anthracis cells. Some agents may form biofilms that enhance
environmental survival, including B. abortus and F. tularensis. In addition to determining persistence
durations under different conditions, the ecological factors contributing to the potential establishment
of an agent in the environment are also important to understand.
This report highlights broad gaps in persistence data. For example, no persistence data for aerosols were
identified for B. anthracis (vegetative cells), Brucella species, B. mallei, B. pseudomallei, or C. burnetii.
Persistence data in soil were lacking for B. anthracis (vegetative cells), B. mallei, F. tularensis, and the
viral encephalitis and hemorrhagic fever agents. Likewise, no persistence data for fomites and water
were identified for C. burnetii.
viii
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1. Introduction
The Centers for Disease Control and Prevention (CDC) categorizes bioterrorism agents based on their
threat to national security. For example, Category A agents are "easily disseminated or transmitted from
person to person" and cause high rates of mortality, while Category B agents are "moderately easy to
disseminate" and cause illness with low mortality (CDC, 2013). Persistence is the ability of an organism
to remain viable (i.e., to remain alive or in the case of viruses to remain infective) over time under a
given environmental condition. Sinclair et al. (2008) conducted a literature review on the persistence of
Category A agents in the environment to help assess risk and guide response actions. As described by
Sinclair et al. (2008), viable agent persistence "will affect decontamination, infection rates, and
encompassing geographic areas. Therefore, knowledge of microbial ecology and defensive public
preparation are important factors in limiting bioterrorism-related morbidity and mortality."
Unfortunately, limited Category A persistence data (e.g., agent die-off rates following a release) were
available at the time of Sinclair's review in 2008.
2. Purpose
The purpose of this study was to determine the state of scientific knowledge about the persistence of
CDC-listed Category A agents since the Sinclair et al. (2008) article was published and to several Category
B agents that were not addressed by Sinclair et al. (2008). These data will allow informed emergency
response and remediation decisions following a contamination incident, and will identify gaps in the
current state of the science so that research can be conducted to close these gaps. The agents that are
addressed in this review are shown in Table 1. While data from attenuated or vaccine strains of agent
were used in this review, only limited surrogate (e.g., same genus but different species than the target
agent) data were considered. With regards to persistence of Bacillus anthracis, only the vegetative (not
spores) form of the organism was considered, as the spore-form is known to persist for decades or
more. In general, much of the persistence work published recently has focused on using the actual
agents rather than surrogates, possibly to reduce the uncertainty of extrapolating results from one
species to another. The scope of this study generally focused on agents that were viable as determined
by culturing on artificial media and/or infecting tissue cultures. In some cases evidence of an agent
entering a viable but nonculturable (VBNC) state is discussed. The ability of an agent to subsequently
cause infection or induce human disease following release into the environment is beyond the scope of
this review. Data from Sinclair et al. (2008) were summarized briefly within this document as applicable.
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Table 1. Agents Investigated
Agent
Bacillus anthracis*
Brucella species (e.g., suis,
melitensis, abortus, etc.)
Burkholderia mallei
Burkholderia pseudomallei
Coxiella burnetii
Francisella tularensis
Viral encephalitis and
hemorrhagic fever agents
Yersinia pestis
Disease
Anthrax
Brucellosis
Glanders
Melioidosis
Q fever
Tularemia
Encephalitis and
hemorrhagic fever
Plague
CDC
Category
A
B
B
B
A
A and B
A
Organism
Type
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Virus
Bacteria
Reviewed by
Sinclair et al. (2008)?
Yes
No
No
No
Yes
Yes
Yes
* This review focused on vegetative B. anthracis only.
3.
Methods
Data for the agents targeted in this literature review (Table 1) were considered from unclassified
reports, peer-reviewed journal articles, and published books. The review focused on the environmental
persistence of the agents associated with aerosols, fomites (i.e., surfaces or materials likely to carry
contamination), soil, and water. The media of interest play potentially important roles in the fate and
transport of the agents, as well as potential media to which humans could be easily exposed. For
example, humans could be exposed to agents via the inhalation of contaminated air, physical contact
with contaminated fomites or soil (or the inhalation or reaerosolized agents from fomites and soil), and
ingestion of contaminated water. The search was limited to articles published in English, but there was
no restriction on geographic location. The literature search for Category A agents previously reviewed by
Sinclair et al. (2008) generally focused on publications from 2007 to 2013. However, relevant articles
identified from earlier time periods were also included in this review if not previously included in the
review by Sinclair et al. (2008). The literature search for Category B agents, which were not reviewed by
Sinclair et al. (2008), was more open to publications from all time periods. Books were limited to those
published or revised in the last five years. The primary search engine used was Google Scholar. Search
terms included the agent name: anthracis, brucella, mallei, pseudomallei, burnetii, tularensis, viral
hemorrhagic fever, filovirus, arenavirus, ebola + marburg, lassa + machupo, viral encephalitis, or pestis.
The search terms were the agent name plus "persistence", agent name plus "recoverable", agent name
plus "survival" and "survivability", and agent name plus "viability", for example:
• anthracis + persistence
• anthracis + recoverable
• anthracis + survival + survivability
• anthracis + viability
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In general, the first 100 returns (sorted by relevance) for each search term were reviewed for potentially
applicable data. Additional search terms were used in conjunction with the agent name plus persistence
approach as a check to ensure important variables affecting the agent's persistence were captured
including: temperature, relative humidity (RH), surface type, agent preparation, associated
environmental matrix, time, and ultraviolet (UV) light or solar radiation. Fewer returns (approximately
20 to 40) were reviewed for the additional searches.
In addition, the U.S. Environmental Protection Agency's (US EPA) Support for Environmental Rapid Risk
Assessment (SERRA) database was used to search literature on persistence for B. anthracis, Brucella
species, B. mallei, B. pseudomallei, F. tularensis, and Y. pestis. Data from the "Fate and Persistence"
node of SERRA was used as applicable. The PubMed database was occasionally used in the literature
review as a check against articles being identified with Google Scholar. As part of the literature review,
potentially useful references in the bibliographies of the obtained documents were reviewed for
relevance and obtained as applicable.
This report was generated using references (secondary data) that could not be evaluated for accuracy,
precision, representativeness, completeness, or comparability and therefore no assurance can be made
that the data extracted from these publications meet the stringent quality assurance requirements of
the US EPA. However, the sources of secondary data were limited to peer-reviewed documents. While
there are no formal quality requirements (professional judgment was used), each article was assessed
qualitatively according to the document evaluation categories and data quality indicators shown in Table
2. These data quality indicators are the same as those used for documents included in US EPA's SERRA
database. The articles cited within this report have generally been incorporated into the SERRA
database, including completed "document criteria reports" that summarize the information presented
in Table 2 for each article. The SERRA database is available at https://serra.cbiac.org/serra/ and requires
registration for an account prior to use.
Table 2. Document Evaluation Categories and Data Quality Indicators
Document Evaluation Category Data Quality Indicators
Literature/Scientific Content A. Primary research publication
Level*
B. Scientific/technical assessment
C. Topical/literature review
D. Opinion article
Statistical Analysis: A- Statistical findings mentioned or presented (summary)
B. Methodology, raw data and statistical findings presented
(comprehensive)
C. Statistics not indicated
D. Not applicable
Technology/Methods Used: A- AM novel technology/methods
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Experimental Methods:
Model Organism(s) Used:
Controls:
B. Some novel technology/methods, some established
technology/methods
C. All established technology/methods
D. Origin of technology/methods unknown
E. Not applicable
A. Included with satisfactory detail
B. Not included
C. Incomplete/missing important information
D. Not applicable
A. No surrogate agent used (original agent used for all
studies)
B. Surrogate agent used for some studies
C. Surrogate agent used for all studies
D. Not applicable
A. Controls used for all studies
B. Controls used for some studies but not others
C. No controls used for any of the studies
D. Controls not indicated/described
E. Not applicable
4. Bacillus anthracis Vegetative Cell Persistence
B. anthracis is a member of the Bacillus cereus group, which is commonly found within soils. However,
unlike the other members of the B. cereus group, B. anthracis can cause three different types of illness
in humans: cutaneous, gastrointestinal, or inhalational anthrax. Robert Koch first described the etiology
of anthrax in 1876 (Koch, 1876). Hudson et al. (2008) has described the history of significant work which
has been devoted to the organism in the years since by Koch, Louis Pasteur, and others, as well as the
use of B. anthracis as a bioweapon. When vegetative B. anthracis cells are exposed to unfavorable
growth conditions, such as contact with air, they rapidly sporulate to form persistent spores (Hudson et
al., 2008). Due to their known prolonged persistence in the soil (Kracalik et al., 2013), B. anthracis spores
have been the focus of many studies, while a lesser amount of work has focused on the vegetative form
of the bacterium. This review focuses only on studies conducted with vegetative cells of B. anthracis. A
separate document focusing on the persistence of B. anthracis vegetative cells and spores in soil is in
process.
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4.1. B. anthracis Vegetative Cells on Fomites
The persistence of vegetative B. anthracis cells is considered to be significantly less than spores on
environmental surfaces because of their nutrient demands, fragile nature, and inability of compete with
other microorganisms (Hugh-Jones and Blackburn, 2009). Therefore, fewer studies have devoted their
focus to vegetative work. This review found only three studies that dealt with the persistence of
vegetative B. anthracis cells or surrogate Bacillus species on different fomites; two of which focused on
the ability of the bacteria to form biofilms within nutrient broth.
Galeano et al. (2003) found that when high concentrations of B. anthracis Sterne vegetative cells,
approximately 10s to 107 colony forming units (CPU) per milliliter (ml), were placed on stainless steel
coupons and held at 25 °C and 80% RH to prevent desiccation, the vegetative cell counts increased over
24 hours. The study also tested antimicrobial-coated stainless steel coupons and found that culturable
vegetative cells began to decrease after only 2 hours of incubation on the treated coupons and were
inactivated by three orders of magnitude by 24 hours (Galeano et al., 2003). Unfortunately, the study
only quantified cell viability up to 24 hours, and thus does not increase our understanding of
persistence on stainless steel over greater temporal scales (i.e., >1 day).
The capability of B. anthracis to form biofilms might have a profound impact on its growth and
persistence on various surfaces. Due to their structure, biofilms are known to be relatively antibiotic
resistant (Lee et al., 2007). Therefore, biofilm microenvironments might create a more suitable
environment for vegetative B. anthracis to flourish. Auger et al. (2009) tested multiple strains of the 6.
anthracis surrogates Bacillus thuringiensis and B. cereus for their ability to form biofilms on polyvinyl
chloride microtiter plates containing lysogeny broth with bactopeptone at 30°C. After 72 hours of
incubation, 47% of the B. thuringiensis and 37.5% to 40% of the nonclinical and diarrheal B. cereus
strains formed biofilms (Auger et al., 2009). In another study, B. anthracis Sterne cells began to form a
biofilm on glass microtiter plates within 8 hours when brain heart infusion (BHI) broth was held under
static conditions, and a mature biofilm was found after 7 days of incubation. In contrast, when the BHI
broth was laminarly flowed within a three-channel flow cell, 24 hours were required for microcolony
formation, yet a mature biofilm was again noted after 7 days (Lee et al., 2007). As these studies show, 6.
anthracis has the capacity to form biofilms under select conditions, and it is possible that the biofilms
may aid in its persistence within the environment.
As mentioned previously, the ability of B. anthracis to form biofilms could have a profound impact on
environmental persistence and proliferation. Biofilm formation protects B. anthracis cultures through a
variety of mechanisms: the multi-layer structure of biofilms serves to protect the inner cells from
potentially hazardous conditions. Biofilms are sloughed off in clumps thereby creating a mechanism for
transportation with embedded protective capabilities. Finally, biofilm formation can create areas of
nutrient limitation, thereby triggering spore formation for the B. anthracis cells (Lee et al., 2007).
However, Lee et al. (2007) determined that the environmental conditions during which the biofilm is
formed impacts the ratio of spores to vegetative cells founds within a mature biofilm. When biofilms
were supplemented with 5% carbon dioxide (CO2) during culture, the vegetative cell concentration was
greater than 88% as compared to less than 50% under normal atmospheric conditions (no
5
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supplemented CO2) (Lee et al., 2007). The work by Lee et al. (2007), as well as Galeano et al. (2003) and
Auger et al. (2009), is summarized in Table 3.
For Table 3, and the other persistence tables presented in this report, the longest persistence duration is
shown for each applicable agent, material, and environmental condition. To better describe the upper
range of persistence, a later column "shortest duration without persistence" is also included. Actual
persistence will likely fall between the durations presented in these columns. For example, Galeano et
al. (2003) tested the persistence of Bacillus species at 0, 2, 6, and 24 hours. When testing was conducted
with stainless steel with an antimicrobial coating, viable cells were recovered at 6 hours but not at 24
hours, so under the tested conditions the bacteria survive between 6 and 24 hours. When testing was
conducted with stainless steel, persistence occurred at the longest duration tested (i.e., a duration was
not tested that failed to recover viable bacteria), and the actual persistence duration could be longer
than 24 hours, which is reflected on Table 3.
Table 3. Bacillus Species (Vegetative Cells) Persistence on Fomites
Bacillus
species
B. cereus T,
B. subtilis
168, and
B. anthracis
Sterne
B. anthracis
Sterne
Material
Stainless
steel
Stainless
steel with an
antimicrobial
coating
Polystyrene
and glass
Environmental
Condition
25°C, 80% RH
25°C, 80% RH
Biofilm growth
in static BHI
Longest
Duration with
Persistence
24 hours*
6 hours
7 days*
Shortest
Duration without
Persistence
-
24 hours
-
Study
Galeano et al.
(2003)
Lee etal. (2007)
B.
thuringiensis
and B.
cereus
broth, 37°C,
5% CO2
Glass Biofilm growth 7 days*
in laminar
flowing BHI
broth, 37°C
Polyvinyl Biofilm growth 3 days*
chloride in lysogeny
broth with
bactopeptone,
30°C
Auger et al.
(2009)
-- Not tested/not reported.
* The longest duration tested (i.e., the actual persistence duration could be longer).
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4.2. B. anthracis Vegetative Cells in Soil
Sinclair et al. (2008) noted that the life cycle of B. anthracis includes a soil dwelling stage and that viable
spores can be recovered after several years (e.g., 40 years based on the work by Manchee et al. [1994]).
However, other than a cursory mention that germination and multiplication may occur within the soil
dwelling stage, no mention as to how, and under what conditions B. anthracis spores germinate within
soil were highlighted within the Sinclair et al. (2008) review. The concept that spores germinate and
propagate within soil is not new. For example, West and Burges (1985) spiked spores of B. thuringiensis
and B. cereus into sandy silt loam soils supplemented with grass clippings or chicken manure. Their
results showed that B. thuringiensis spores germinated and propagated within the grass-supplemented
soil, but B. cereus did not. After 24 days, the B. thuringiensis counts came to a plateau and remained at
22 times the spore inoculum level for the duration of the experiment, while B. cereus had an initial
germination spike then declined to 0.11 times the inoculum. B. thuringiensis and B. cereus spores
germinated within the manure-supplemented soil; however, after the initial burst of activity the viability
counts decreased to 0.22 times the inoculum value for B. thuringiensis and to 0.098 times the inoculum
for B. cereus (West and Burges, 1985). These results indicate that though spores may germinate when
an abundance of fresh nutrients are available within supplemented soils, the germinated cells do not
readily persist in the manure-soil environment (West and Burges, 1985). Others works have suggested
that B. thuringiensis can proliferate within vegetation. Tilquin et al. (2008) found that B. thuringiensis
subspecies israelensis spores, originally sprayed in French lands as a means of mosquito control, were
found in high concentrations within leaf matter (3 x 10s spores per gram [g]). The authors suggest that
the high concentrations of B. thuringiensis found within the decaying leaf litter may be because the leaf
litter is a specific microenvironment in which the organism can persistence and grow. Specifically the
work targeted two key elements to the leaf litter microenvironment that potentially contributed to the
seen increase in spore counts- low oxygen levels and low decomposition rates (Tilquin et al., 2008).
While these studies did not utilize vegetative B. anthracis, their work is important because both 6.
thuringiensis and B. cereus spores are routinely used as surrogates for B. anthracis spores. A limited
number of studies have also focused on the ability of B. anthracis to propagate within soil environments.
In one such work, B. anthracis spores were found to germinate and propagate within the rhizosphere of
a common pasture grass (Saile and Koehler, 2006). The authors proposed that the plant roots enhanced
the germination and proliferation of vegetative cells, as nearly 50% of the inoculated spores germinated
in the presence of plant roots, where as little to no spores germinated in the absence of the grass (Saile
and Koehler, 2006). The study also found evidence of the plasmid pBC16, which confers tetracycline
resistance, being transferred between two strains of B. anthracis within their model rhizosphere system.
This finding is significant as it provides strong evidence of metabolically active B. anthracis cells within
the plant-soil environment (Saile and Koehler, 2006).
A series of studies have focused on the interaction between worms (nematodes or earthworms) and
Bacillus species in both their vegetative and spore forms. Laaberki and Dworkin (2008) fed B. anthracis
Sterne in Nematode Minimal Media agar (spores) or Nematode Growth Medium plates (vegetative cells)
to laboratory-controlled nematodes. They found that the digestive track of the worm killed the
consumed vegetative cells, but spores were able to pass unaffected through their system into their
7
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feces. This conclusion is supported by work conducted by Hendriksen and Hansen (2002). However,
Hendriksen and Hansen (2002) were able to further deduce that 6. thuringiensis spores consumed with
soil germinated within the gut of the earthworm prior to resporulation and defecation. Furthermore, the
study showed that B. thuringiensis was able to germinate within the hindgut of three of the four tested
earthworm species, indicating that this is not a species limited occurrence, but rather a widely
distributed condition (Hendriksen and Hansen, 2002). More recently, the interaction between
earthworms and 6. anthracis has been shown to be dependent upon the presence of various
bacteriophages (viruses that infect bacteria). The bacteriophages appear to induce various phenotypic
changes to the B. anthracis vegetative cells that alter their capacity "to sporulate, produce
exopolysaccharide, form biofilms, and survive long-term in soil" (Schuch and Fischetti, 2009). Multiple
studies have shown that only bacteriophage-infected B. anthracis are capable of infecting the intestinal
tract of earthworms, and that the bacteriophage present are variable (Schuch and Fischetti, 2009;
Schuch et al., 2010). Schuch et al. (2010) showed that one bacteriophage, Wipl, was present in high
numbers for three years within a Pennsylvania forest floor soil before the population was dramatically
replaced by a second, Wip4, bacteriophage for the next three years of the study. This demonstrates that
even the bacteriophage population within environmental B. anthracis-\\ke isolates is variable due to any
number of environmental factors potentially including the composition of the soil that they are
ingesting.
Another point of interest is the comparison of the earthworm lifestyle to what is known regarding the 6.
anthracis spore lifecycle within soils. Both earthworms and B. anthracis spores prefer alkaline soils with
high calcium levels and rich in organic matter (Hugh-Jones and Blackburn, 2009; Schuch et al., 2010).
Anthrax events also seem to occur more commonly after seasonal flooding events when earthworms
retreat to the soil surface to escape the water-saturated ground (Schuch et al., 2010). The combination
of lifestyle patterns, hindgut colonization, and anthrax occurrence patterns points to a significant
relationship between anthrax occurrence and earthworms. While direct sampling at enzootic areas
would be required to definitively determine a correlation between earthworms and anthrax outbreaks,
their overlapping lifestyles suggests that B. anthracis spores are carried upward to the soil surface and
potentially onto vegetation via colonized earthworm digestive systems.
4.3. B. anthracis Vegetative Cells in Water
Sinclair et al. (2008) presented data from Busson (1911) and Mitscherlich and Marth (1984) indicating
that vegetative B. anthracis persists for 72 hours to 6 days in water. Since Sinclair et al. (2008), only one
study was identified that looked at environmental waters. Dey et al. (2012) utilized a low nutrient creek
water to assess the interaction of B. anthracis with Acanthamoeba castellanii, a common soil amoeba.
The work clearly showed a pathway where ingested B. anthracis spores germinate within the amoeba
and replicate to the point of lysing the amoeba host. Upon lysis, the vegetative cells then sporulated in
the low nutrient creek water. Virulent B. anthracis contains two plasmids, pXOl and pX02. Both plasmids
must be present for virulence; pXOlcodes for the exotoxin and pX02 codes for encapsulation.
Interestingly, a pXOl plasmid dependence was noted for spore germination within the amoeba. The
8
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researchers hypothesized that this germination dependence may explain why pX02 is lacking in multiple
natural B. anthracis strains - it is not required for proliferation (Dey et al., 2012).
4.4. B. anthracis Vegetative Cell Persistence Gaps
Within this literature search, no specific data on the persistence of aerosolized 6. anthracis vegetative
cells were found. Furthermore, little data were found regarding their persistence on various surfaces.
There seems to be some indication that cells can proliferate for a short period of time (e.g. 24 hours;
Galeano et al., 2003); however, to date the open literature does not provide data on extended periods
of incubation, or a full understanding of when and where proliferation occurs. In addition, testing of
vegetative cells on fomites was not conducted at environmental conditions <25°C, <80% RH, or with
simulated sunlight. Sagripanti et al. (2007) conducted a systematic decontamination analysis of multiple
Bacillus species spores on various materials of interest. US EPA (2010a) has also conducted a study
looking at the persistence of 6. anthracis spores on wood, glass, concrete, and soil in simulated sunlight.
Future work utilizing similar experimental designs as either Sagripanti et al. (2007) or US EPA (2010a)
with vegetative cells would shed significant light on the overall persistence of 6. anthracis cells. Studies
have also shown that 6. anthracis is capable of forming biofilms on glass and polyvinyl chloride surfaces
when covered with nutrient media, however the required conditions and the overall persistence of the
formed biofilms have yet to be explored (Auger et al., 2009; Lee et al., 2007).
The interaction between soil and water species (e.g., amoebae, bacteriophages, earthworms, and
grasses) with 6. anthracis have been brought to the forefront in recent studies (Dey et al., 2012;
Hendriksen and Hansen, 2002; Laaberki and Dworkin, 2008; Saile and Koehler, 2006; Schuch and
Fischetti, 2009; Schuch et al., 2010). However, there remain gaps within the overall understanding of
how the various species influence 6. anthracis proliferation within soil or water. The studies included
herein have shown that 6. anthracis can colonize the hindgut of earthworms and infect amoeba,
however questions remain on if this colonization actually increases spore numbers within the
surrounding environment. There also remains questions regarding the virulence of the 6. anthracis
spores if/when they do persist. Dey et al. (2012) found that only the pXOl plasmid was required for
spore germination within amoeba, and may account for why pX02 is often lacking in environmental
strains. However, it is unknown if 6. anthracis can regain virulence with the uptake of the pX02 plasmid
in the environment. A clear linkage between the various soil or waterborne species and the known 6.
anthracis geographic distribution patterns of 6. anthracis as presented by Blackburn et al. (2007) and
Griffin et al. (2009) could strengthen the epidemiological connection between soils and anthrax
incidence.
5. Brucella species Persistence
There are several closely related species of Brucella that are each associated with a different host. For
example, Brucella melitensis is typically associated with goats, Brucella suis is associated with swine, and
Brucella abortus is often associated with cattle (Franz et al., 1997). All three of these Brucella species are
9
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capable of infecting humans (Franz et al., 1997), and are CDC-listed agents. Accordingly, persistence data
were gathered for these three Brucella species. Persistence data were not identified for other species of
Brucella.
5.1. Brucella species on Fomites
Three studies were identified that evaluated the persistence of B. suis on fomites, which are
summarized in the following paragraphs and Table 4. These studies were based on the inoculation of 107
to 10s CPU of B. suis onto the material coupons. The material coupons were sterilized by autoclaving or
gamma irradiation prior to inoculation with B. suis. Fomite persistence studies evaluating Brucella
species other than B. suis were not identified.
10
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Table 4. B. suis (ATCC 23444) Persistence on Fomites
Material
Aluminum
Carpet
Keyboard keys
Painted joint tape
Aluminum
Concrete
Glass
Soil
Wood
Aluminum
Glass
Wood
Environmental Condition
22"C, 35% RH
22"C, 35% RH
22°C, 45% RH
22°C, 45% RH
22°C, 40% RH
22°C, 50% RH
5°C, 10% RH
5°C, 60% RH,
22"C, 40% RH
22°C, 50% RH
5°C, 50% RH
5"C, 50% RH,
22"C, 40% RH
22"C, 50% RH
5°C, 10% RH
5"C, 50% RH,
22"C, 40% RH
22°C, 50% RH
5"C, 10% RH
5°C, 60% RH,
22°C, 40% RH
5°C, 10% RH
22°C, 40% RH
5°C, 30% RH
22°C, 40% RH
5°C, 30% RH
22°C, 40% RH
5°C, 30% RH
, simulated sunlight
simulated sunlight
, simulated sunlight
simulated sunlight
, simulated sunlight
simulated sunlight
, simulated sunlight
simulated sunlight
Longest Duration
with Persistence
7 days*
7 days*
7 days*
4 hours
28 days*
7 days
28 days*
5 days
<7 days
<1 day
7 days
<1 day
28 days*
Iday
28 days*
2 days
28 days*
14 days*
28 days*
14 days*
<21days
28 days*
56 days*
56 days*
56 days*
56 days*
42 days
56 days*
Shortest Duration _ .
without Persistence ^
Ryan (2010)
J
-
8 hours
USEPA(2010b)
10 days
-
5 days
7 days
Iday
14 days
Iday
-
2 days
-
2 days
-
-
-
-
21 days
-
Calfee and Wendling
" (2012)
-
B
56 days
-
-- Not tested/not reported.
* The longest duration tested (i.e., the actual persistence duration could be longer).
11
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The US EPA conducted persistence testing with 6. suis following inoculation onto aluminum, carpet,
keyboard keys, and painted joint tape (Ryan, 2010). Briefly, material coupons were inoculated with 1 x
107 viable B. suis in 100 microliter (ul) aliquots of stock suspension (BHI broth). The inoculated coupons
were held at approximately 35% RH or 45% RH and 22°C. At various time points, the coupons were
placed in phosphate buffered saline (PBS) and agitated on an orbital shaker to extract the 6. suis from
the materials. Aliquots of the undiluted extract and associated serial dilutions were spread plated onto
BHI agar and incubated for up to 72 hours at 37°C. 6. suis was found to persist on aluminum, carpet, and
keyboard keys for at least 7 days (the longest duration tested). On painted joint tape (surrogate for
painted wallboard), B. suis was recovered after 4 hours but not after 8 hours.
Similar persistence testing with B. suis was conducted by US EPA (2010b) using surfaces of aluminum,
concrete, glass, soil, and, on a limited basis, wood. Testing was conducted at two temperatures
(approximately 5°C and 22°C) with and without exposure to UV radiation intended to mimic sunlight.
The tests were conducted under ambient/uncontrolled RH that generally averaged from 40% to 60% RH,
although the long term testing at 5°C without UV averaged about 10% RH. In the absence of UV, B. suis
persisted for at least 28 days on aluminum, glass, and soil at 5°C and 22°C, for 7 days on concrete at 5°C
(although persistence on concrete was <7 days at 22°C), and for 28 days on wood at 5°C (although
persistence on wood was <21 days at 22°C). Persistence was decreased in the presence of UV. B. suis
was recovered from aluminum after 5 days at 5°C and after 7 days at 22°C, from glass after 2 days at 5°C
and after 1 day at 22°C, from soil after 14 days at 5°C and 22°C when exposed to UV. B. suis persisted <1
day on concrete when exposed to UV at 5°C and 22°C. B. suis persistence on wood was not tested under
simulated sunlight. B. suis appeared to generally persist better at 5°C than 22°C (US EPA, 2010b).
Even longer term B. suis persistence testing (56 days) was reported by Calfee and Wendling (2012) on
glass, aluminum, and wood. Testing was conducted at two environmental conditions: 22°C and 40% RH,
and 5°C and 30% RH. B. suis persisted on aluminum and glass under both environmental conditions for
at least 56 days. B. suis also persisted on wood for 56 days at 5°C and persisted for approximately 42
days at 22°C. B. suis persistence was higher at 5°C than 22°C (Calfee and Wendling, 2012).
5.2. Brucella species in Soil
Brucella species can persist for several weeks in soil (Franz et al., 1997; Charters, 1980) and in dust
(Franz et al., 1997). Nicoletti (1980) briefly summarized the persistence results of B. abortus in soil. The
cited data were either incomplete or from foreign language publications. As a result, the details of the
persistence studies summarized by Nicoletti (1980) were limited. Nevertheless, B. abortus appeared to
survive better in moist soil than dry soil, as the bacterium persisted 66 days in wet soil, persisted 48 to
73 days in soil at 90% humidity, and persisted <4 days in dried soil (Nicoletti, 1980). As shown in Table 4
for fomites, B. suis can persist at least 28 days (the longest duration tested) in soil held at 5°C or 22°C
(US EPA, 2010b).
Jones et al. (2010) noted that sunlight and elevated temperatures impact B. abortus survival, such that
persistence in the environment is expected to be a few weeks in Yellowstone National Park during the
12
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summer. More specifically, Aune et al. (2012) found that B. abortus can persist on fetal tissues, soil, or
vegetation in the Greater Yellowstone Area for several weeks depending upon the month, temperature,
and sunlight. For example, the persistence of B. abortus applied to fetal tissues (bovine and bison) held
in shaded areas was measured twice weekly. Fetuses deployed in February, March, April, and May had
B. abortus persistence of 81, 77, 69, and 25 days, respectively (Aune et al., 2012). Soil and vegetation
sampling at naturally occurring B. abortus contaminated sites associated with bison births or abortions
was also conducted weekly. B. abortus persisted 10 to 43 days at sites identified in April and persisted
for 7 to 26 days at sites identified in May (Aune et al., 2012).
5.3. Brucella species in Water
Franz et al. (1997) reported that Brucella species can survive for several weeks in water, but the bacteria
are sensitive to heat. (Franz et al. [1997] did not specify the temperatures associated with heat
sensitivity.) A literature review by Nicoletti (1980) found data indicating that B. abortus in water
survived >57 days at 8°C, 77 days at room temperature, and <1 days at 37°C. Many of the specific details
surrounding the persistence of B. abortus in the environment were not summarized by Nicoletti (1980).
Falenski et al. (2011) inoculated mineral water (held at 20°C) with B. abortus (strain 1119-3) at 5 x 107
CFU ml"1; Brucella colonies were detected after 60 days but not after 63 days.
Gilbert and Rose (2012) used autoclaved, dechlorinated municipal water inoculated with 10s CFU ml"1 6.
melitensis or B. suis to assess persistence at 5°C and 25°C. Both Brucella species were culturable
between 1-2 days at 25°C and between 7-9 days at 5°C. In addition to culturing, viability was assessed by
measuring metabolic (i.e., esterase) activity. Based on esterase production, both Brucella species
remained viable for 30 days (the longest study duration) at 5°C and 25°C, suggesting that the bacteria
entered a VBNC state. The persistence data from Nicoletti (1980), Falenski et al. (2011), and Gilbert and
Rose (2012) are summarized in Table 5.
13
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Table 5. Bruce/la Species Persistence in Water
Brucella
species
B. abortus
B. abortus
(1119-3)
B. melitensis
(ATCC 23456)
B. suis
(EAM 562)
Longest Duration Shortest Duration
Environmental Condition Study
with Persistence without Persistence
Room temperature
Lake water, 8°C, pH 6.5
Lake water, 37°C, pH 7.5
Mineral water, 20°C
Autoclaved, dechlorinated
municipal water, 5°C
Autoclaved, dechlorinated
municipal water, 25°C
Autoclaved, dechlorinated
municipal water, 5°C
Autoclaved, dechlorinated
municipal water, 25°C
77 days* Nicoletti (1980)
>57 days*
<1 day*
Falenskietal. (2011)
60 days 63 days
Gilbert and Rose
7 days 9 days
(2012)
1 day 2 days
7 days 9 days
1 day 2 days
-- Not tested/not reported.
* Data obtained from a literature review, the shortest duration without persistence was not reported.
5.4. Brucella species Persistence Gaps
Specific data on the survival of Brucella species in aerosols were not identified. However, Brucella
species appears to be adversely affected by warmer temperatures and exposure to sunlight (e.g., Jones
et al., 2010). On fomites, Brucella species persistence varied by surface type (e.g., Ryan, 2010). It is
uncertain if material-specific interactions adversely affected recovery from some surfaces (e.g., painted
joint tape and concrete). Recent evidence indicates that Brucella species may enter a VBNC state (e.g.,
Gilbert and Rose, 2012). The persistence of B. melitensis and B. suis in water (<9 days at 5°C and 25°C)
reported by Gilbert and Rose (2012) was considerably lower than the persistence of B. abortus in water
(>57 days at 8°C and 20°C) reported by Nicoletti (1980) and Falenski et al. (2011), respectively. It is
uncertain if the difference is attributed to species-level differences or the study parameters. Under
certain conditions (e.g., nutritionally deficient, low oxygen) B. abortus was found to aggregate and
produce biofilms, which appear to enhance the bacteria's tolerance to desiccation (Almiron et al., 2013).
Additional research is needed on the mechanisms that contribute to the environmental persistence of
Brucella species including species-specific data. For example, for fomites, only persistence data
associated with B. suis were identified.
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6. Burkholderia mallei Persistence
6. mallei is primarily associated with horses and is not expected to survive outside its host for long
durations (Gilad et al., 2007; Dvorak and Spickler, 2008). Dvorak and Spickler (2008) reviewed data
indicating that B. mallei might survive in wet, humid, or dark conditions for 3 to 5 weeks, but survival is
reduced in the presence of sunlight. Specific data relative to the limited survival of B. mallei outside the
host including the environmental media or surface types associated with persistence were not provided.
6.1. B. mallei on Fomites
Specific studies designed to generate persistence data of B. mallei on fomites were not identified.
However, Malik et al. (2012) stated that 6. mallei "has an affinity for warm and moist conditions and
may survive for up to 3 months in stable bedding, manure, feed and water troughs (particularly if
heated), wastewater and equine transporters (saddler and harness equipment)". Specific data
supporting these statements were not provided by Malik et al. (2012), but the statement seems to
support the environmental conditions more suitable for B. mallei persistence noted by Dvorak and
Spickler (2008).
6.2. B. mallei in Water
Three studies were identified investigating the persistence of B. mallei in water (Table 6). Miller et al.
(1948) reported that B. mallei inoculated into tap water survived 4 weeks at room temperature, but 6.
mallei was not recovered after 5 weeks. Moore et al. (2008) inoculated sterile distilled, deionized water
with B. mallei (approximately 10s CPU ml"1) and found that the bacterium was culturable after
approximately 27 days but not culturable after 31 days (this persistence duration was estimated from
Figure 1 of Moore et al. [2008]). Gilbert and Rose (2012) used autoclaved, dechlorinated municipal
water inoculated with 10s CPU ml"1 6. mallei to assess persistence at 5°C and 25°C. 6. mallei was found
to be viable by culturing for only for 1 to 2 days when held at 25°C or 5°C. In addition to culturing,
viability was assessed by measuring metabolic (i.e., esterase) activity. Based on esterase production, 6.
mallei remained viable for 30 days (the longest study duration) at 5°C and 25°C, suggesting that the
bacterium remained alive but was unable to be recovered by culturing on an artificial medium.
15
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Table 6. B. mallei Persistence in Water
B. mallei
Isolate
Not specified
ATCC 23344
M-13
Longest Duration
Environmental Condition
with Persistence
Tap water, room
temperature
Sterile distilled, deionized
water
Autoclaved, dechlorinated
municipal water, 5°C
Autoclaved, dechlorinated
municipal water, 25°C
4 weeks
27 days
Iday
Iday
Shortest Duration
Study
without Persistence
5 weeks
31 days
2 days
2 days
Miller etal. (1948)
Moore etal. (2008)
Gilbert and Rose
(2012)
6.3. B. mallei Persistence Gaps
With the exception of survival data in water, relatively little persistence data were identified for 6.
mallei. Specific data on the survival of B. mallei in aerosols or on soil were not identified. The
information on B. mallei persistence on fomites was not supported with specific data or laboratory
controlled studies. Although three persistence studies were identified with B. mallei in water, the results
were somewhat conflicting. Gilbert and Rose (2012) observed considerably shorter durations of
culturability than Miller et al. (1948) or Moore et al. (2008). It is unknown what caused such different
persistence results or why B. mallei entered a VBNC state during the Gilbert and Rose (2012) study.
16
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7. Burkholderia pseudomallei Persistence
7.1. B. pseudomallei on Fomites
One study focused on the persistence of B. pseudomallei on fomites. Shams et al. (2007) investigated
the survival of 6. pseudomallei (suspended in Butterfield buffer or BHI broth) applied to glass, stainless
steel, paper, and polyethylene. Testing was conducted with two 6. pseudomallei isolates (ATCC 11668
and 23343). Test temperatures and RH were not specified in Shams et al. (2007). Viability was assessed
using culture methods and a solid-phase cytometerto detect esterase activity. When analyzed by
culture and applied in Butterfield buffer, both isolates of 6. pseudomallei persisted between 6 and 24
hours on all materials, except isolate ATCC 11668, which persisted between 24 hours and 3 days on
paper (Table 7). When analyzed by culture and applied in BHI broth, both isolates of 6. pseudomallei
persisted between 24 hours and 3 days on glass and persisted between 3 and 7 days on paper (Table 7).
When applied to polyethylene in BHI broth, the ATCC 11668 isolate persisted between 24 hours and 3
days, and the ATCC 23343 isolate persisted between 3 and 7 days (Table 7). When applied to stainless
steel in BHI broth, the ATCC 11668 isolate persisted between 3 and 7 days, and the ATCC 23343 isolate
persisted between 6 and 24 hours (Table 7). When assessed by esterase activity (not shown in Table 7),
6. pseudomallei generally persisted at least 14 days when applied in Butterfield buffer and persisted at
least 35 days when applied in BHI broth. The measure of esterase activity detected longer persistence,
possibly because the bacteria entered a VBNC state or because the cells were injured. Persistence
appeared to increase when 6. pseudomallei was suspended in BHI broth, which is a more complex
medium with slower drying than Butterfield buffer.
17
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Table 7. B. pseudomallei Persistence on Fomites from Shams et al. (2007)
B. pseudomallei Material
Isolate
ATCC 11668 Glass
ATCC 23343
ATCC 11668
ATCC 23343
ATCC 11668 Paper
ATCC 23343
ATCC 11668
ATCC 23343
ATCC 11668 Polyethylene
ATCC 23343
ATCC 11668
ATCC 23343
ATCC 11668 Stainless steel
ATCC 23343
ATCC 11668
ATCC 23343
Application
Suspension
Butterfield buffer
Butterfield buffer
BHI broth
BHI broth
Butterfield buffer
Butterfield buffer
BHI broth
BHI broth
Butterfield buffer
Butterfield buffer
BHI broth
BHI broth
Butterfield buffer
Butterfield buffer
BHI broth
BHI broth
Longest Duration
with Persistence
6 hours
6 hours
24 hours
24 hours
24 hours
6 hours
3 days
3 days
6 hours
6 hours
24 hours
3 days
6 hours
6 hours
3 days
6 hours
Shortest Duration
without Persistence
24 hours
24 hours
3 days
3 days
3 days
24 hours
7 days
7 days
24 hours
24 hours
3 days
7 days
24 hours
24 hours
7 days
24 hours
7.2. B. pseudomallei in Soil
As reviewed by Dance (2000), B. pseudomallei occurs in tropical and sub-tropical climates and is
associated with decaying organic matter in the environment. While B. pseudomallei is infrequently
found in surface soil (the organism may be adversely affected by sunlight), subsurface samples (e.g., at a
25-60 centimeter [cm] depth) are more likely to yield 6. pseudomallei (Dance, 2000). 6. pseudomallei is a
facultative anaerobe (Reckseidler et al., 2001) and can grow in anoxic environments (Dance, 2000).
Laboratory studies have demonstrated long survival durations in soil at room temperature, although
some B. pseudomallei strains survive months in the laboratory at 5°C (Dance, 2000). B. pseudomallei has
been recovered from soil with a wide range in pH, but the bacterium may prefer slightly acidic
conditions (Dance, 2000). More detailed data on the persistence of B. pseudomallei in soil are provided
in the following paragraphs and Table 8.
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Table 8. B. pseudomallei Persistence in Soil
B. pseudomallei Isolate
Environmental Condition
Longest Duration
with Persistence
Shortest Duration
without Persistence
Study
9 isolates (unspecified) from soil
collected from a sheep paddock in
Australia
8 isolates (H35, H43, H47, G106, G123,
G139, Gdl54, and Gdl73) from water
and soil in China (Hainan, Guangxi, and
Guangdong)
12 isolates (1 through 12) from 12
melioidosis patients in Taiwan
Isolate (unspecified) from soil collected
near an ephemeral creek in Australia
Isolate (unspecified) from soil collected
near an ephemeral creek in Australia
KN07
Initially moist soil stored in plastic bags
on a shaded shelf in a laboratory at
ambient temperature (13°C to 33°C)
0% water content at room temperature
5% water content at room temperature
10% water content at room temperature
20% water content at room temperature
40% water content at room temperature
80% water content at room temperature
5% water content
10% water content
15% water content
20% water content
Soil dried (<0.1% moisture), after 104
days the moisture was adjusted to 15%
Soil intermittently wetted (9.9%
moisture), after 104 days the moisture
was adjusted to 15%
Sterile soil inoculated with 6.
multivorans, B. pseudomallei, and other
soil bacteria
30 months
(other soil samples
tested had shorter
durations)
30 dayst
40 dayst
70 dayst
439 dayst
726 days*
726 days*
16 days
50 days
150 days*
150 days*
113 days*
113 days*
<10 days
36 months
(other soil samples
tested had shorter
durations)
Thomas and
Forbes-Faulkner
(1981)
Tongetal. (1996)
20 days
70 days
Chen et al. (2003)
Larsenetal. (2013)
10 days
Lin etal. (2011)
-- Not tested/not reported.
* The longest duration tested (i.e., the actual persistence duration could be longer).
t Although apparently tested, the shortest duration without persistence was not reported.
19
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As described by Thomas and Forbes-Faulkner (1981), Australian soil samples were collected from a-
fenced area with sheep and then stored in plastic bags on a shaded shelf in a laboratory at ambient
temperature (13°Cto 33°C). Attempts at B. pseudomallei isolation by culture were made at 6-month
intervals. B. pseudomallei was found to survive up to 30 months in soil (described as a moist medium
clay originally collected from a soil depth of 40-45 cm).
B. pseudomallei appears to benefit from moist soil, but may persist in soil that gradually dries. long et
al. (1996) reported survival times of 40, 70, 439, and at least 726 days in soil with water contents of 5%,
10%, 20%, and 40%, respectively. Chen et al. (2003) similarly observed that B. pseudomallei survived
approximately 16 to 20 days in soil with a water content of 5%, approximately 50 to 70 days in soil with
10% water, and at least 150 days in soil with 15% to 20% water. Larsen et al. (2013) inoculated 6.
pseudomallei into sterile soil to study the influence of soil moisture on survival. B. pseudomallei was not
recovered following inoculation into dry soil. However, B. pseudomallei persisted in initially moist soil
(approximately 10% moisture) that underwent drying for at least 91 days and survived 113 days (the
longest duration tested) when intermittently irrigated with sterile distilled water. Larsen et al. (2013)
concluded that dry endemic soil may act as a reservoir for B. pseudomallei during the dry season.
Based on testing at 4°C, 22°C, 25°C, 30°C, 37°C, 42°C, and 45°C, Chen et al. (2003) reported that 37°C to
42°C were optimal temperatures for B. pseudomallei growth in soil, although some strains were able to
grow at 4°C. Chen et al. (2003) also reported that the optimal soil pH was 6.5 to 7.5 (based on testing at
pH values of 3.5, 4, 4.5, 5.5, 6.5, 7.5, 8, and 8.5), although 2 of 12 strains survived at pH 4 in soil.
Although not specifically tested in soil, long et al. (1996) reported optimal survival conditions for 6.
pseudomallei to be temperatures of 24°C to 32°C (based on testing at 0°C, 8°C, 16°C, 24°C, 32°C, 40°C,
and 48°C) and pH ranging from 5 to 8 (based on testing at pH values of 2, 3, 4, 5-8, 9, and 10). In normal
saline (the salt content was undefined), eight strains of B. pseudomallei averaged 18 days survival at 0°C
and averaged 28 days survival at 40°C (long et al., 1996). long et al. (1996) also reported that 6.
pseudomallei were killed by radiation from an UV lamp (465 microwatt [uW]/square centimeter [cm2]
for 7.75 minutes), but noted that the radiation associated with natural sunlight can be absorbed by
other materials thereby limiting sunlight's killing effect.
Studies have been conducted in Thailand to determine soil properties associated with the presence of 6.
pseudomallei. Palasatien et al. (2008) found that B. pseudomallei was primarily associated with sandy
soil, at a depth of 30 cm, pH of 5.0 to 6.0, >10% moisture, and elevated total nitrogen and chemical
oxygen demand relative to soils without recoverable B. pseudomallei (Palasatien et al., 2008). Palasatien
et al. (2008) noted that under conditions of low water content, organic matter in the soil may enable 6.
pseudomallei to survive. Suebrasri et al. (2013) evaluated Thailand soil properties associated with 6.
pseudomallei during the rainy season and dry season. A higher prevalence of B. pseudomallei positive
locations (22%) occurred during the rainy season compared with 6% positive locations during the dry
season. During the rainy season, three soil properties were identified as being different between
positive and negative sites. Locations with B. pseudomallei had a mean soil pH of 6.05, a percentage of
water holding capacity of 31.92%, and an iron concentration of 16.33 milligram per kilogram (mg kg"1).
Locations negative for B. pseudomallei had a mean soil pH of 5.51, a percentage of water holding
capacity of 40.17%, and an iron concentration of 109.86 mg kg"1. The percentage of water holding
20
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capacity is a function of soil composition with sandy soils having a lower water holding capacity than
soils comprised of silt and clay. During the dry season only the manganese content was identified as
being different between B. pseudomallei positive sites (138.46 mg kg"1) and negative sites
(46.88 mg kg"1) (Suebrasri et al., 2013). The authors discussed the possibility that B. pseudomallei
entered a VBNC state to survive the dry season (Suebrasri et al., 2013). Despite the statistical differences
between some of the physiochemical properties of soil at B. pseudomallei positive and negative sites,
scatter plots shown by Suebrasri et al. (2013) (but not shown in this report) demonstrate nearly
complete overlap between the results at positive and negative sites. Additional research is needed to
understand the influence of soil characteristics on the persistence of B. pseudomallei.
Based on soil samples (collected at a depth of 30 cm) from northern Australia, Kaestli et al. (2009) found
that the environmental parameters associated with the presence of B. pseudomallei differed depending
upon undisturbed and manipulated (e.g., farmed) areas. In undisturbed areas, B. pseudomallei was
associated with closeness to streams, moist soil, grassy areas, and areas associated with native animals
(e.g., wallabies). In manipulated areas, B. pseudomallei was associated with the presence of livestock,
clay loam soil (i.e., a clay, silt, and sand mixture), red brown (an indication of oxidized iron) clay soil, and
lower (5.5) soil pH. Kaestli et al. (2009) also noted that B. pseudomallei was recovered from dry soil in
manipulated areas suggesting that other factors, besides water, support B. pseudomallei growth. Later
research by Kaestli et al. (2012) found that B. pseudomallei was associated with the rhizosphere, roots,
and above ground parts of various grasses, especially non-native grasses introduced for grazing animals.
Lin et al. (2011) demonstrated that other soil bacteria (e.g., Burkholderia multivorans) could inhibit 6.
pseudomallei growth. The authors inoculated sterile soil with B. multivorans, B. pseudomallei, and other
soil bacteria. Cultures resembling B. multivorans were observed 10 days after inoculation, but 6.
pseudomallei was not observed. Polymerase chain reaction (PCR) techniques were able to detect 6.
pseudomallei DNA 20 days after inoculation but not after 30 days.
As reviewed by Inglis and Sagripanti (2006), B. pseudomallei can form biofilms and survive in amoeba
cysts and fungi, which may be important aspects of B. pseudomallei in the environment. For example,
Levy et al. (2009) detected B. pseudomallei genetic material in fungal spores collected from soil samples.
The soil samples were not culture positive for B. pseudomallei. Levy et al. (2009) speculated that the
association of the bacteria with fungal spores might contribute to B. pseudomallei persistence and
dispersal in the environment.
7.3. B. pseudomallei in Water
Several studies have documented B. pseudomallei survival in distilled and environmental waters (Table
9). Moore et al. (2008) reported that B. pseudomallei survived in sterile distilled, deionized water for at
least 200 days. Wuthiekanun et al. (1995) reported that B. pseudomallei persisted at least 3 years in
sterile distilled water without any additional nutrients; the ambient temperature varied from 25°C to
40°C. Pumpuang et al. (2011) noted that B. pseudomallei persisted for 16 years in distilled water held at
25°C.
21
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Table 9. B. pseudomallei Persistence in Water
B. pseudomallei Isolate
1026b
Soil isolate (unspecified)
207a
NCTC 10276, NCTC 13177, and BCC 11
NCTC 10276
NCTC 13 177
BCC 11
NCTC 13 177
Environmental Condition
Sterile distilled, deionized water
Sterile distilled water, ambient
temperature 25°C to 40°C
Distilled water, 25°C
Filter-sterilized rain water
Sterile distilled water, 40°C
Sterile distilled water, 20°C
Sterile distilled water, 2°C
Sterile distilled water, pH 7
Sterile distilled water, pH 6
Sterile distilled water, pH 5
Sterile distilled water, pH 4
Sterile distilled water, pH 3
Sterile distilled water, pH 3
Sterile distilled water, pH 3
Water with an artificial sea salt
concentration of 0.004%
Water with an artificial sea salt
concentration of 0.04%
Water with an artificial sea salt
rrtnr^ntratirm rtf O A.0/*
Longest Duration
with Persistence
200 days*
3 years*
16 years*
28 days*
28 days*
28 days*
28 days*
28 days*
28 days*
28 days*
28 days*
5 days
7 days
2 days
28 days*
28 days*
28 days*
Shortest Duration
without Persistence
Moore etal. (2008)
Wuthiekanun et al.
(1995)
Pumpuang et al.
(2011)
Robertson et al.
(2010)
—
-
—
7 days
14 days
5 days
22
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B. pseudomallei Isolate Environmental Condition
Water with an artificial sea salt
concentration of 4%
Water with an artificial sea salt
concentration of 40%
12 isolates (1 through 12) from Autoclaved pond water
melioidosis patients in Taiwan Autoclaved river water
Autoclaved estuary water
Autoclaved sea water
ATCC 23343 Autoclaved, dechlorinated
municipal water, 5°C
Autoclaved, dechlorinated
municipal water, 25°C
NCTC 13177 Artificial sea water exposed to
sunlight
Sterile distilled water exposed
to sunlight
Rain water exposed to sunlight
Longest Duration
with Persistence
28 days*
Iday
150 days*
150 days*
20 days
8 days
Iday
30 days*
60 minutes
60 minutes
60 minutes
Shortest Duration
without Persistence
-
2 days
::
36 days
12 days
2 days
90 minutes
90 minutes
90 minutes
Study
Chen etal. (2003)
Gilbert and Rose
(2012)
Sagripanti et al.
(2009)
-- Not tested/not reported.
* The longest duration tested (i.e., the actual persistence duration could be Ion
ger).
23
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6. pseudomallei persistence in sterile distilled water, rain water (sterilized by filtration), and artificial sea
water was studied for 28 days (Robertson et al., 2010). 6. pseudomallei survived 28 days in distilled
water and rain water. More specifically, B. pseudomallei survived 28 days in distilled water at 40°C, 20°C,
and 2°C. B. pseudomallei also survived 28 days in distilled water at pH levels of 7', 6, 5, and 4. In distilled
water at a pH level of 3, B. pseudomallei persistence varied by isolate (i.e., 5 to 7 days for NCTC 10276, 7
to 14 days for NCTC 13177, and 2 to 5 days for BCC 11), although epifluorescent microscopy identified a
few viable cells after 28 days. B. pseudomallei survived 28 days in water with artificial sea salt
concentrations of 0.004% to 4%; however, at a 40% artificial sea salt concentration, B. pseudomallei only
persisted 1 to 2 days. The various sea salt concentrations were prepared using an artificial seawater
base that was reconstituted with demineralized hypo-osmolar water and then autoclaved (Robertson et
al., 2010).
Chen et al. (2003) observed long term B. pseudomallei survival (150 days, the longest duration tested) in
water collected from ponds and rivers, but much shorter persistence occurred in estuarine water
(persisting approximately 20 days but <36 days) and seawater (persisting approximately 8 days but <12
days).
Gilbert and Rose (2012) used autoclaved, dechlorinated municipal water inoculated with 6.
pseudomallei to assess persistence at 5°C and 25°C. B. pseudomallei was culturable for 30 days (the
longest duration tested) at 25°C and was culturable between 1-2 days at 5°C. Metabolic (i.e., esterase)
activity was also measured using solid-phase cytometry to determine viability. Based on esterase
production, B. pseudomallei remained viable for 30 days at 25°C and 5°C. These results suggest that 6.
pseudomallei entered a VBNC state at 5°C (Gilbert and Rose, 2012).
A study of unchlorinated groundwater (rural water supplies) in northern Australia, found that positive 6.
pseudomallei water samples were primarily associated with acidic water (e.g., pH of 6.3 to 6.8), low
water hardness (e.g., 25 to 100 mg per liter [L"1]), low salinity (e.g., 0.02 to 0.07 millisiemens [mS] cm"1),
and higher iron levels (e.g., 2 to 4 mg L"1) than samples negative for B. pseudomallei (Draper et al., 2010).
Sagripanti et al. (2009) studied the survival of B. pseudomallei in water (sterile distilled water, artificial
seawater, and rainwater) exposed to sunlight at Perth, Australia via UV transparent dishes held on ice. In
all three water types, the bacteria survived 60 minutes, but the bacteria were not culturable at 90
minutes. Sagripanti et al. (2009) also investigated the impact of sunlight on B. pseudomallei internalized
by amoeba, which resulted in similar B. pseudomallei persistence as when exposed in water only (i.e., no
apparent protection from sunlight was provided to B. pseudomallei internalized by amoeba).
7.4. B. pseudomallei Persistence Gaps
6. pseudomallei can be transported by aerosols (Sprague and Neubauer, 2004), and 6. pseudomallei has
been isolated from aerator spray associated with a water treatment plant (Inglis et al., 2000). However,
specific data on the survival durations of B. pseudomallei in aerosols were not identified. Only one study
(Shams et al., 2007) was identified that investigated the persistence of B. pseudomallei on fomites. The
24
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influence of different environmental conditions (temperature and humidity) was not investigated as part
of the fomite persistence study. In general, the measured persistence of 6. pseudomallei may be
affected by strain (e.g., long et al., 1996), physiologic variability associated with gene regulation (e.g.,
Larsen et al., 2013), inoculation suspension (e.g., Shams et al., 2007), and method to assess survival
(e.g., Shams etal., 2007).
With regard to 6. pseudomallei persistence in soil and water, Dance (2000) noted that environmental
factors dictating the occurrence of B. pseudomallei in the environment are not well established. Inglis
and Sagripanti (2006) similarly noted that although B. pseudomallei is associated with water and soil, it
is not uniformly dispersed in the environment and "data on its preferred microhabitat are lacking".
Moore et al. (2008) reported that the molecular survival mechanisms for B. pseudomallei in water are
not understood. Moore et al. (2008) noted several factors that enable the environmental persistence of
B. pseudomallei including "a versatile metabolic capacity which allows the use of a variety of carbon
sources for growth, the ability to live inside other microorganisms including protozoa and fungi and the
ability to tolerate a wide range of environmental conditions including a variety of soils and water
environments".
8. Coxiella burnetii Persistence
The bacterium C. burnetii is an obligate intracellular organism that develops spore-like forms resistant to
environmental stressors (Arricau-Bouvery and Rodolakis, 2005). The bacterium may exist as "large-cell
variants (LCV), small-cell variants (SCV), and small dense cells (SDC)" physiological forms, with SCV and
SDC being the environmentally persistent forms (Arricau-Bouvery and Rodolakis, 2005). C. burnetii is not
known to be overly affected by high or low temperatures, drought, or humidity levels (Aitken et al.,
1987). The stability of SCV can theoretically result in environmental persistence for years after being
shed from infected animals (Kersh et al., 2013). C. burnetii can invade a variety of hosts including
amoebae, ticks, birds, and mammals that likely allow the bacterium to be disseminated throughout the
environment (Arricau-Bouvery and Rodolakis, 2005).
8.1. C. burnetii in Aerosols
"The environmental survival of C. burnetii allows it to be transported by wind far away from its original
source" (Arricau-Bouvery and Rodolakis, 2005). Welsh et al. (1958) inoculated pregnant sheep with C.
burnetii and then sampled the air of the sheep's pens for the bacterium. C. burnetii was found in the air
9 to 14 days (the longest duration sampled) after parturition. Welsh et al. (1958) acknowledged that
some cross-contamination in air samples likely occurred even though the sheep were housed in
individual cubicles. In addition, ongoing sources of contamination (e.g., infected sheep feces) might have
contributed to the air contamination. That is, detection of viable C. burnetii in individual cubicles may
have been due to cross contamination or may have been caused by ongoing sources of contamination -
infected sheep feces, rather than actual persistence of the bacterium after parturition (Welsh et al.,
1958). Kersh et al. (2013) detected C. burnetii DNA in the air of a goat farm, which was associated with a
25
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Q fever outbreak one year earlier. Quantitative estimates of persistence from such data are difficult to
make given the potential for re-introduction of the bacteria into the sampled areas.
8.2. C. burnetii on Fomites
Welsh et al. (1958) indicated that "epidemiological evidence exists that C. burnetii may be carried over
considerable distances on inanimate objects, such as clothing, wool, hair, straw, packing materials, and
dust." The authors did not document the durations or environmental condition that C. burnetii may
remain viable. Kersh et al. (2013) detected C. burnetii DNA in high-efficiency particulate air (HEPA)
vacuum samples from the floors and furniture of a farmhouse associated with a Q fever outbreak one
year earlier. During a Q fever outbreak in 2008, de Bruin et al. (2011) used quantitative PCRto detect C.
burnetii DNA at affected farms. Interestingly, more copies of the target DNA sequence were detected
from dusty environmental surfaces than from veterinary samples. As concluded by de Bruin et al. (2011),
the sampling of surfaces that have accumulated dust reflect the occurrence of C. burnetii over a
relatively long time period, while the veterinary samples provide an indication of C. burnetii shedding at
that moment only. The authors acknowledged that the quantitative PCR does not give an indication of
the bacterium's viability. C. burnetii is a difficult organism to culture given its "virulence and complicated
growth requirements" (de Bruin et al., 2011).
8.3. C. burnetii in Soil
Evstigneeva et al. (2007) inoculated C. burnetii into various peat and loamy soils, which were then held
at 20°C, 4°C, and -20°C. The bacterium's survival was determined by inoculation of soil liquids into mice
and monitoring the immunofluorescence of the mice spleens. Results were based on the number of C.
burnetii in the field of vision and the brightness of their fluorescence. C. burnetii survived for 20 days
(the longest duration tested) in all soil types and temperatures (Evstigneeva et al., 2007). Properties of
the soils studied at the three temperatures included: hygroscopic moisture ranged from 1.14% to 7.04%,
pH ranged from 6.10 to 7.75, organic carbon ranged from 1.28% to 14.26%, and the particle-size
composition for <0.01 millimeter (mm) particles ranged from 29.6% to 40.9% and for <0.001 mm
particles ranged from 10.3% to 23.2% (Evstigneeva et al., 2007).
Welsh et al. (1959) periodically sampled soil from ranches with C. burnetii infected sheep. Soil was
collected from lamb birthing areas from one lambing season until the next lambing season. C. burnetii
was present in the soil for up to 150 days, although it is possible that reinfection occurred at the sample
locations (Welsh et al., 1959). Kersh et al. (2013) detected C. burnetii DNA in the soil of a goat farm,
which was associated with a Q fever outbreak one year earlier.
La Scola and Raoult (2001) found that C. burnetii was able to survive within amoeba (A. castellanii) in a
laboratory study. Maurin and Raoult (1999) reported that C. burnetii inside amoeba survived for 6
weeks. La Scola and Raoult (2001) concluded that soil amoeba could provide an intracellular niche for
the survival of C. burnetii in a spore-like form.
26
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8.4. C. burnetii Persistence Gaps
Data on the persistence of C. burnetii in water were not specifically identified. Much of the persistence
data of C. burnetii (in aerosols, on fomites, and some soil studies) were based on environmental
sampling associated with Qfever outbreaks. These studies often used PCR techniques that did not
assess the organism viability nor were the studies conducted in controlled laboratory settings, so
recontamination could have occurred. Arricau-Bouvery and Rodolakis (2005) noted that "the
physiological forms of [C. burnetii] excreted in milk, feces or placentas are unknown. The form excreted
by the host and the hypothetical possibility that LCV could convert to SCV in the environment are crucial
information for implementing adequate strategies for the disinfection of feces or parturition products
that could directly contaminate humans or the environment."
9. Frandsella tularensis Persistence
F. tularensis (including the tularensis, holarctica, and novicida subspecies) is the causative agent of
tularemia. Based on the review by Hazlett and Cirillo (2009), the bacteria are capable of surviving in
diverse environments and can infect mammals, arthropods, and protozoans. For example, F. tularensis
subspecies tularensis is found in rabbits and rodents of North America with transmission occurring via
ticks and bloodsucking flies (e.g., horseflies). The tularensis subspecies has not traditionally been known
to be associated with an aquatic reservoir. However, F. tularensis subspecies holarctica, which is found
more widely in the northern hemisphere, is associated with water-borne disease and is transmitted by
mosquitoes, ticks, and biting flies (Hazlett and Cirillo, 2009). F. tularensis subspecies novicida, sometimes
referred to as F. novicida, is generally less virulent than other F. tularensis subspecies and "is associated
with water-borne transmission" (Durham-Colleran et al., 2010). "The endemic nature of Frandsella
across the northern hemisphere suggests environmental persistence most probably utilizing numerous
mechanisms" (Mahajan et al., 2011).
A study was conducted on the distribution of dog ticks (Dermacentor variabilis) infected with F.
tularensis subspecies tularensis, which is believed to be responsible for the sustained outbreak of
tularemia on the island of Martha's Vineyard, Massachusetts (Goethert and Telford, 2009). A small niche
(approximately 290 meters in diameter) was identified where F. tularensis is perpetuated and genetic
diversity is generated, although the authors noted that there was nothing obvious in the habitat to
enhance persistence or transmission (Goethert and Telford, 2009). In discussing pneumonic tularemia
on Martha's Vineyard in 1978 and 2000, Feldman et al. (2001) noted that the patients were likely
exposed on the southern coast where closeness to brackish ponds and ocean-related precipitation might
contribute to F. tularensis persistence. Interestingly, the F. fr//arens/s-infected ticks analyzed by Goethert
and Telford (2009) were collected from the southern coast near Squibnocket. Later work by Berrada and
Telford (2011) suggested that F. tularensis subspecies tularensis persists on Martha's Vineyard because
of sulfur/salt-influenced water or soil that is conducive to F. tularensis growth.
Nakazawa et al. (2010) modeled the ecological niche of F. tularensis in the United States at the
subspecies (tularensis and holarctica) and clade (Al, A2, Ala, Alb of the tularensis subspecies) level.
27
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Coarse-scale (e.g., county-level) ecological parameters were used to identify potentially distinct niches.
Variables in the model included measures of days with frost, temperature, precipitation, solar radiation,
and topographical characteristics. The model predicted that the tularensis and holarctica subspecies
distributions actually overlap much of the country except tularensis is predicted to be absent from some
northern/northeastern states and holarctica is predicted to be absent from five southern states. At the
Al and A2 clade level, predicted distributions were dramatically different with Al occurring in the
central to southeastern states and A2 occurring in the western states. Relative to Al strains, A2 strains
were predicted at higher elevations, with less precipitation, lower temperatures, and more days with
frost. Differences in the predicted distributions of clades Ala and Alb were not apparent.
The above mentioned studies indicate that F. tularensis does persist in the environment, but specific
reservoirs and the role of hosts remain elusive. More specific research is summarized below that
investigated the persistence of F. tularensis in various environmental media.
9.1. F. tularensis in Aerosols
Sinclair et al. (2008) summarized three studies (Cox, 1971; Cox and Goldberg, 1972; Ehrlich and Miller,
1973) from the 1970s on the persistence of F. tularensis aerosols and estimated times required for the
initial titer to decrease by 99% (T99 values). Based on the 24 T99 values (e.g., 0.48 hours [29 minutes
following wet dissemination at 50% RH] to 131 hours [5 days following wet dissemination at 90% RH])
estimated by Sinclair et al. (2008), aerosolized F. tularensis is expected to possibly persist for several
minutes to a few days. However, other than the T99 value of 131 hours, all T99 values were <7.93 hours
indicating that F. tularensis is most likely to persist <1 day in an aerosol. Sinclair et al. (2008) did not
comment specifically on the uniqueness of the T99 value of 131 hours, which was estimated from
relatively short study durations (e.g., aerosol ages of 15 minutes by Cox [1971] and Cox and Goldberg
[1972]) as were many of the other T99 values. The oldest aerosol age for which F. tularensis data were
evaluated by Sinclair et al. (2008) was a 64-minute study duration reported by Ehrlich and Miller (1973).
One F. tularensis persistence article (Hood, 2009) for aerosols that had been published since the review
by Sinclair et al. (2008) was identified. Hood (2009) studied the persistence of F. tularensis strain Schu S4
in aerosols (particle sizes of <3 micrometers [fim, 3 (im to 6 (im, and >6 (im) held at 9°C to 13°C and 72%
to 85% RH, and found persistence after 45 minutes to 60 minutes (the longest durations tested). These
tests were conducted in a highly ventilated system such that the exposures were equivalent to open air
exposures. Interestingly, the author noted that unidentified constituents in the air (possibly olefins from
oil refineries and dense car populations) may have reduced F. tularensis viability (i.e., lowered the
percent survival). The viability of F. tularensis <3 (im particles after 45 minute exposures ranged from
10% to 80% survival depending on the wind direction associated with the air intake during testing. In
addition, the viability of F. tularensis <3 (im particles was reduced in the ventilated system but viability
was not reduced in comparable testing conducted with a non-ventilated system (Hood, 2009).
28
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9.2. F. tularensis on Fomites
Persistence testing was conducted with F. tularensis live vaccine strain (LVS) following inoculation onto
aluminum, carpet, keyboard keys, and painted joint tape (Ryan, 2010). Briefly, material coupons were
inoculated with 1 x 107 viable F. tularensis in 100 ul aliquots of stock suspension (Muller-Hinton broth).
The inoculated coupons were held at approximately 35% RH and 23°C. At various time points, the
coupons were placed in PBS and agitated on an orbital shaker to extract the F. tularensis from the
materials. Aliquots of the undiluted extract and associated serial dilutions were spread plated onto
chocolate II agar plus ISoVitaleX™ ( Becton, Dickinson and Company, Franklin Lakes, NJ) and incubated
for up to 72 hours at 37°C. F. tularensis was found to persist (although the mean recovery was <1%) on
the keyboard key material after 7 days (the longest duration tested).The longest F. tularensis persistence
(also <1% mean recoveries) on the other materials was 8 hours on aluminum and painted joint tape and
4 hours on carpet.
Faith et al. (2012) reported that F. tularensis LVS (100-300 CFU) applied to filter paper and allowed to
desiccate survived between 20 and 40 minutes. Only one study (Wilkinson, 1966) with data on F.
tularensis persistence on fomites was identified by Sinclair et al. (2008). Wilkinson (1966) used aerosol
deposition of F. tularensis LVS onto stainless steel. At 25°C, F. tularensis persisted approximately 15 days
at 10% RH, 3 days at 65% RH, and 2 days at 100% RH, as determined via culture. At 37°C F. tularensis
persistence decreased much more rapidly (e.g., within 16 hours at 55% to 100% RH), although under
conditions of 0% RH F. tularensis remained viable for 16 days.
As summarized on Table 10, Wilkinson (1966) and Ryan (2010) found that on some materials (i.e.,
keyboard keys or stainless steel) F. tularensis is capable of surviving for several days at 23°C to 25°C.
Shorter persistence (<24 hours) was observed at warmer temperatures (37°C) by Wilkinson (1966) or on
other materials (aluminum, carpet, and painted joint tape) by Ryan (2010). At 23°C and 35% RH, Ryan
(2010) recovered F. tularensis LVS after 8 hours on aluminum and painted joint tape (but not after 3
days) and after 4 hours on carpet (but not after 8 hours). Wilkinson (1966) observed increased
persistence with decreased humidity including persistence of F. tularensis for at least 16 days under hot
and dry conditions. Faith et al. (2012), however, found rapid loss of viability within 40 minutes under
conditions of desiccation. Faith et al. (2012) used considerably lower inoculums levels (100 to 300 CFU)
than Wilkinson (1966) (107 CFU), and the bacteria used by Faith et al. (2012) were washed and
resuspended in PBS. Wilkinson (1966) used F. tularensis aerosolized along with growth media, such that
the bacteria were likely coated with an outer layer of non-living material, which could have contributed
to the longer persistence.
29
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Table 10. F. tularensis LVS Persistence on Fomites
Material
Stainless steel
Aluminum
Carpet
Keyboard keys
Painted joint tape
Filter paper
Environmental
Condition
25°C, 10% RH
25°C, 65% RH
25°C, 100% RH
37°C, 0% RH
37°C, 55% RH
37°C, 65% RH
37°C, 80% RH
37°C, 100% RH
23°C, 35% RH
23°C, 35% RH
23°C, 35% RH
23°C, 35% RH
Desiccated
Longest Duration
with Persistence
15 days*
3 days*
2 days*
16 days*
16 hours*
12 hours*
12 hours*
9 hours*
8 hours
4 hours
7 days*
8 hours
20 minutes
Shortest Duration
without Persistence
-
-
-
-
-
-
-
-
3 days
8 hours
-
3 days
40 minutes
Study
Wilkinson (1966)
Ryan (2010)
Faith etal. (2012)
— Not tested/not reported.
* The longest duration tested (i.e., the actual persistence duration could be longer).
9.3. F. tularensis in Water
Water Temperature and Salinity
Two studies were identified that evaluated the survival of F. tularensis in water at different
temperatures or salinities. One study (Gilbert and Rose, 2012) found no persistence after 1 day in de-
chlorinated municipal water at 5°C and 25°C, but persistence of at least 14 days at 8°C. A second study
(Berrada and Telford, 2011) found relatively long survival at 21°C in brackish water. The salt and sulfur
content of the brackish water may have contributed to the high survival reported by Berrada and
Telford) (2011) compared to the municipal water used by Gilbert and Rose (2012). More details on these
studies are provided in the following paragraphs.
Gilbert and Rose (2012) used autoclaved, dechlorinated municipal water inoculated with 10s CFU ml"1 F.
tularensis LVS and NY98, which was then held at 5°C, 8°C, or 25°C. Both F. tularensis LVS and NY98 are
holarctica subspecies (US EPA, 2012). Viability was assessed by culture on chocolate II agar at 35°C.
When held at 8°C, F. tularensis LVS was culturable after 14 days (but not after 21 days) and F. tularensis
NY98 was culturable after 28 days (but not after 30 days). F. tularensis LVS and NY98 were not culturable
from water held at 5°C or 25°C after 1 day (i.e., the shortest duration tested without persistence). The
authors note that F. tularensis may have entered into a VBNC state at 5°C and 25°C.
30
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Berrada and Telford (2011) studied the persistence of F. tularensis subspecies tularensis (SSTR9 10-7), F.
tularensis LVS, and F. novicida U112 in freshwater, brackish-water, and saline (0.85% sodium chloride)
when stored at room temperature (21°C). The salinity of the freshwater and brackish-water samples was
not reported, but the sodium levels were 10 mg/L and 3,604 mg/L, respectively. Culturability was
assessed on cystine heart agar blood (CHAB) supplemented with 8% rabbit blood and antibiotics with
incubation at 37°C. Inoculum levels were >107 CPU ml"1. In freshwater, F. tularensis subspecies tularensis
(SSTR9 10-7) and F. tularensis LVS were culturable after 10 days, but not after 14 days, and F. novicida
U112 was culturable after 7 days, but not after 10 days. Persistence was longer in brackish-water as F.
tularensis subspecies tularensis (SSTR9 10-7) and F. novicida U112 were culturable after 28 days, but not
after 34 days, and F. tularensis LVS was culturable after34 days (the longest duration tested). In saline, F.
tularensis LVS and F. novicida U112 were culturable after 18 days, but not after 21 days, and F. tularensis
subspecies tularensis (SSTR9 10-7) was culturable after 21 days, but not after 28 days. The authors noted
that the brackish-water samples contained much higher sulfur residues (168-219 mg mL"1) than the
freshwater samples (2-5 mg L"1), which might enhance F. tularensis survival. In fact, "sulfur-containing
amino acids, cysteine or cystine, are usually required for the cultivation of F. tularensis" (Berrada and
Telford, 2011). Similarly, 1% sodium chloride was previously reported as enhancing F. tularensis culture
growth (Berrada and Telford, 2011).
Sinclair et al. (2008) summarized two studies (DeArmon et al., 1962; Forsman et al., 2000) evaluating the
persistence of F. tularensis. An environmentally relevant study of F. tularensis persistence in tap water
at 8°C (Forsman et al., 2000) was associated with a T99 value of 33.7 days (Sinclair et al., 2008). Forsman
et al. (2000) noted that F. tularensis in water can enter a VBNC state. The other T99 values reported by
Sinclair et al. (2008) were less applicable for environmental scenarios as the studies focused on the
shelf-life of cultures specifically prepared (e.g., packaged in polyethylene ampoules or freeze-dried) for
long-term storage (DeArmon et al., 1962).
Nutrients and Protozoa
Three studies were identified that found associations with high nutrient and/or protozoan levels with F.
tularensis survival in water. Broman et al. (2011) collected surface water samples (e.g., lakes and rivers)
from tularemia endemic areas (i.e., the cities Ljusdal and Orebro) in Sweden. The aquatic systems
sampled could be characterized as eutrophic. F. tularensis subspecies holarctica was detected in water
"during three consecutive years, indicating that the bacterium may persist in water for several years"
(Broman etal., 2011).
Thelaus et al. (2009) investigated the persistence of F. tularensis subspecies holarctica in the laboratory
with lake water. The lake water originated from Lake Nydala in northern Sweden and was filtered and
autoclaved prior to use in the experiment. The lake water was then seeded with 6.0 x 10s F. tularensis
cells mL"1) under differing nutrient and predation conditions. Water samples (1 mL) were subsequently
sampled and bead-beaten in an attempt to release intracellular bacteria, spread on selective modified
Thayer-Martin agar, and incubated at 37°C for 4 days. The bacteria persisted 1 day but were unable to
be cultured after 5 days in a high-nutrient condition (total nitrogen 3.0 mg L"1 and total phosphorus 0.4
mg L"1) with protozoa (Euglenozoa, Cryptomonas, and Ochromonas). Under high nutrient conditions
(without protozoa) or under low-nutrient conditions (total nitrogen 0.3 mg L"1 and total phosphorus
31
-------
0.008 mg L"1) with protozoa (Euglenozoa, Cryptomonas, Ochromonas, Strombilidium, Paramecium, and
Urothrica), F. tularensis were culturable after 16 days, but not after 26 days. Under low-nutrient
conditions without protozoa, F. tularensis were culturable after 26 days, but not after 38 days.
Interestingly, microscopic counts of F. tularensis labeled with green fluorescent protein (gfp) were
obtained over the entire 38-day experiment without significant effects observed for nutrient levels or
predation, although over the first 16 days Francisella-gfp abundance increased under the high nutrient
conditions with protozoa. F. tularensis cells were observed as planktonic as well as inside the protozoa,
which was most efficiently determined by detection of g/p-produced fluorescence. F. tularensis ingested
by the protozoa were not rapidly degraded, and F. tularensis could escape from the protozoa. The
inability to grow on culture plates indicates that the F. tularensis cells had entered a VBNC state. The
inability to culture F. tularensis may be indicative of symbiotic bacteria that obtain proteins from the
host cell (e.g., the protozoa) and down-regulate the synthesis of proteins, possibly losing the ability to
replicate outside the host. Thelaus et al. (2009) also reported that F. tularensis lost its virulence in mice
after incubation in the lake water (after 120 days), irrespective of treatment. Apparently, F. tularensis
can survive and reproduce in water for relatively long periods of time (including waters with high
nutrient levels and protozoan predation), although virulence may be lost.
EI-Etr et al. (2009) indicated that amoeba may be important with regard to the environmental
persistence of F. tularensis. The amoeba, A. castellanii, was shown to rapidly encyst in response to F.
tularensis infection, and virulent strains of F. tularensis were shown to survive in the cysts for at least 3
weeks post-infection (EI-Etr et al., 2009). The authors discuss the possibility that F. tularensis in amoeba
cysts enables the bacteria to survive drying and other adverse environmental conditions.
Biofilms
The ability of F. tularensis strains to form biofilms may be important for its persistence in the
environment. F. tularensis subspecies tularensis strains SchuS4 and FT-10, F. tularensis LVS, and F.
tularensis subspecies novicida form biofilms on abiotic surfaces such as glass (Margolis et al., 2010).
Margolis et al. (2010) reported persistence durations of at least 7 days for F. tularensis subspecies
novicida on chitin-containing surfaces, at least 5 days for F. tularensis subspecies novicida under flow
conditions, at least 6.3 days for F. tularensis LVS and F. tularensis subspecies novicida under static
conditions, and at least 1 day for F. tularensis subspecies tularensis strains SchuS4 and FT-10 under static
conditions (Table 11). Durham-Colleran et al. (2010) demonstrated that F. tularensis subspecies novicida
could produce biofilms (and persist for at least 2 days) at 25°C and 37°C on polystyrene (and on
polyvinyl-chloride at 37°C) when grown in tryptic soy broth supplemented with cysteine. Mahajan et al.
(2011) also demonstrated that F. tularensis LVS can form biofilms in moderately hard water at 37°C and
persist for at least 15 days without nutrients or host cells. Durham-Colleran et al. (2010) concluded that
"by forming a biofilm in natural ecosystems, the nutritionally fastidious Francisella may be able to
survive the environmental conditions of mud and waterways. In addition, biofilm formation could be a
mechanism Francisella utilizes for persistence within the tick arthropod vector."
32
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Table 11. F. tularensis Persistence in Water
F. tularensis
Subspecies and Strain
holarctica LVS
holarctica NY98
holarctica LVS
holarctica NY98
holarctica LVS
holarctica NY98
tularensis SSTR9 10-7
holarctica LVS
novicida U112
tularensis SSTR9 10-7
holarctica LVS
novicida U112
tularensis SSTR9 10-7
holarctica LVS
novicida U112
holarctica LVS
holarctica
holarctica
holarctica
Environmental Condition
Autoclaved, dechlorinated municipal water, 5°C
Autoclaved, dechlorinated municipal water, 5°C
Autoclaved, dechlorinated municipal water, 8°C
Autoclaved, dechlorinated municipal water, 8°C
Autoclaved, dechlorinated municipal water, 25°C
Autoclaved, dechlorinated municipal water, 25°C
Filter-sterilized freshwater, 21°C
Filter-sterilized freshwater, 21°C
Filter-sterilized freshwater, 21°C
Filter-sterilized brackish-water, 21°C
Filter-sterilized brackish-water, 21°C
Filter-sterilized brackish-water, 21°C
Filter-sterilized saline, 21°C
Filter-sterilized saline, 21°C
Filter-sterilized saline, 21°C
T99 value in tap water, 8°C
Lake water with bacteria predators (protozoa) and
high nutrient conditions
Lake water with bacteria predators (protozoa) and
low nutrient conditions
Lake water without bacteria predators and high
nutrient conditions
Longest Duration
with Persistence
<1 day
<1 day
14 days
28 days
<1 day
<1 day
10 days
10 days
7 days
28 days
34 days*
28 days
21 days
18 days
18 days
33.7dayst
Iday
16 days
16 days
Shortest Duration
without Persistence
Iday
Iday
21 days
30 days
Iday
Iday
14 days
14 days
10 days
34 days
-
34 days
28 days
21 days
21 days
-
5 days
26 days
26 days
Study
Gilbert and Rose
(2012)
Berrada and Telford
(2011)
Sinclair etal. (2008)
Thelausetal. (2009)
33
-------
F. tularensis
Subspecies and Strain
holarctica
novicida
novicida
novicida and
holarctica LVS
tularensis SchuS4 and
FT-10
Environmental Condition
Lake water without bacteria predators and low
nutrient conditions
Biofilm growth on chitin-containing surfaces, in
Chamberlain's defined medium without glucose,
30°C
Biofilm growth under flow conditions (0.1 mL
minute'1), 20°C to 22°C
Biofilm growth under static conditions, 26°C and
37°C
Biofilm growth under static conditions, 37°C
Longest Duration
with Persistence
26 days
7 days*
5 days*
6.3 days*
Iday*
Shortest Duration
Study
without Persistence
38 days
Margolis et al.
(2010)
-
-
-
novicida
Biofilm growth on polyvinyl-chloride in tryptic soy
broth supplemented with cysteine, 37°C
-- Not tested/not reported.
* The longest duration tested (i.e., the actual persistence duration could be longer).
t Data obtained from a literature review, the shortest duration without persistence was not reported.
Durham-Colleran et
al.(2010)
novicida
holarctica LVS
LJI *_/ 1 1 1 i gi *_/ vv ii i *_/ii ksuiy-Jiyi^ii*^ 111 n y M i.i\s j\s y ksi *_/ n i
supplemented with cysteine, 25°C and 37°C
Biofilm growth in moderately hard water, 37°C
2 days*
15 days*
—
Mahajan et al.
/ ~)ni 1 \
(2011)
34
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The ability of F. tularensis species to adhere to chitin-containing surfaces was investigated by incubating
F. tularensis subspecies novicida with crab shell (Margolis et al., 2010). After 1 hour at 30°C, bacteria
were observed adhered to the shell surface. After 1 week, bacterial communities were present
consisting of individual bacteria surrounded by a matrix of extracellular polymeric substance. The
observed community structure suggests that F. tularensis subspecies novicida can attach to and
proliferate as biofilms on chitin, an environmentally relevant surface including copepod and zooplankton
shells and exoskeletons of arthropods. The authors also note that F. tularensis subspecies novicida likely
uses chitinases for the hydrolysis of chitin providing a local nutrient source for F. tularensis subspecies
novicida persistence and growth. Data on the persistence of F. tularensis in water are summarized in
Table 11.
9.4. F. tularensis Persistence Gaps
This review of the literature published since Sinclair et al. (2008) did identify additional studies focused
on the persistence of F. tularensis on in aerosols, on fomites, and in water, but no more recent studies
were identified assessing persistence in soil, or studying the influence of solar radiation (UV light) on F.
tularensis persistence.
Many of the studies reviewed attempted to characterize the environmental reservoir of F. tularensis. F.
tularensis does exist in some aquatic and soil environments, and evidence is accumulating that the
natural reservoirs may include protozoa and/or biofilms. The mechanism by which F. tularensis persists
or establishes an environmental reservoir following a release is unknown, but bacterial survival may be
associated with high nutrient content, brackish/saline environments, protozoan predation, biofilms, and
relatively high sulfur or cystine content.
10. Viral Encephalitis and Hemorrhagic Fever Agents Persistence
Category A viral hemorrhagic fever agents include filoviruses (e.g., Ebola and Marburg) and arenaviruses
(e.g., Lassa and Machupo) (CDC, 2013). The Sinclair et al. (2008) review identified persistence data on
the Marburg virus and Lassa virus. Sinclair et al. (2008) also identified data for flaviviruses (Japanese
encephalitis virus, St. Louis encephalitis virus, and yellow fever virus), bunyaviruses (hantavirus and
Crimean-Congo virus), and alphaviruses (Venezuelan equine encephalitis [or encephalomyelitis] [VEE]
virus). Alphaviruses (e.g., VEE) causing viral encephalitis is an example of a Category B agent, and
hantavirus is an example of a Category C agent (an emerging infectious disease) (CDC, 2013).
10.1. Viral Encephalitis and Hemorrhagic Fever Agents in Aerosols
In aerosols, Sinclair et al. (2008) reported that the Marburg virus was not persistent in air (based on data
from Belanov et al., 1996). Sinclair et al. (2008) estimated T99 values of 1.42 to 3.58 hours for the Lassa
virus, 5.97 to 11.2 hours for the Japanese encephalitis virus, 2.32 to 291 hours [12.1 days] for the VEE
35
-------
virus, and 9.58 to 2,875 hours [119.8 days] for the St. Louis encephalitis virus. Although T99 values were
not estimated, hantavirus is reportedly stable in air (Schmaljohn and Hjelle, 1997 as cited in Sinclair et
al., 2008). Based on data presented by Sinclair et al. (2008), the Japanese encephalitis virus and VEE
virus appeared to have inverse relationships with RH. The highest T99 values for the St. Louis encephalitis
virus (2,875 hours and 51.1 hours were associated with the lowest RH levels tested (23% and 35%,
respectively). The other T99 values for the St. Louis encephalitis virus ranged from 9.28 to 25.4 hours
over RH levels of 46% to 80% without any apparent trends.
Piercy et al. (2010) studied the persistence of Ebola and Marburg viruses in aerosols held at
approximately 50% RH and 22°C. The viruses could be detected (i.e., infect cell cultures) after 90
minutes (the longest duration tested) in the aerosol. Smither et al. (2011) used spiders' webs to capture
aerosolized Ebola and Marburg viruses as an alternative method of assessing persistence. Smither et al.
(2011) reported that both viruses survived 60 minutes (the longest duration tested) when held at
approximately 50% RH and 22°C, similar to the results obtained using the more dynamic aerosol system
of Piercy et al. (2010). The available persistence data identified for viral encephalitis and hemorrhagic
fever agents in aerosols is also summarized in Table 12.
Table 12. Viral Encephalitis and Hemorrhagic Fever Agents Persistence in Aerosols
Virus
St. Louis
encephalitis virus
VEE virus
Lassa virus
Japanese
encephalitis virus
Ebola virus
Marburg virus
Ebola virus
Marburg virus
Environmental
Condition
21°C, 23% RH
9.0°C, 19% RH
32°C, 80% RH
24°C, 30% RH
22°C, 50% RH
22°C, 50% RH
22°C, 50% RH
22°C, 50% RH
Longest Duration
with Persistence
119.8 dayst
(T99 value)
12.1 dayst
(T99 value)
3.58hourst
(T99 value)
11.2 hourst
(T99 value)
1.5 hours*
1.5 hours*
1 hour*
1 hours*
Shortest Duration
without Persistence
Sinclair et al. (2008)
-
-
-
Piercy et al. (2010)
-
Smither etal. (2011)
-
-- Not tested/not reported.
* The longest duration tested (i.e., the actual persistence duration could be longer).
t Data obtained from a literature review, the shortest duration without persistence was not reported.
36
-------
10.2. Viral Encephalitis and Hemorrhagic Fever Agents on Fomites
Sinclair et al. (2008) found data (from Belanov et al., 1996) indicating that the Marburg virus was stable
in dried blood for 4 to 5 days. Bunyaviruses (Crimean-Congo virus and hantavirus, which can cause
hemorrhagic fever) were estimated to have T99 values ranging from 2.16 to 2.91 hours, respectively on
aluminum (Sinclair et al., 2008, based on research conducted by Hardestam et al., 2007). The
persistence data identified for viral encephalitis and hemorrhagic fever agents on fomites is summarized
in the following text and Table 13.
Fogarty et al. (2008) reported that the half-lives for henipaviruses (e.g., Hendra virus and Nipah virus,
which can cause encephalitis) on fruit (mango) flesh from 12 minutes to 30.3 hours, depending on
temperature (generally longer persistence at 22°Cthan 37°C) and fruit pH (longer persistence at pH 5
than pH 4.5 or 3.5). Desiccation of the viruses within a polystyrene dish resulted in Hendra virus and
Nipah virus survival for <15 minutes when stored at 37°C, Hendra virus survival for 30 minutes (but not 1
hour) when stored at 22°C, and Nipah virus survival for 1 hour (the longest duration tested) when stored
at 22°C (Fogarty et al., 2008).
37
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Table 13. Viral Encephalitis and Hemorrhagic Fever Agents Persistence on Fomites
Virus
Hantavirus
Crimean-Congo
virus
Marburg virus
Hendra virus
Hendra virus
Nipah virus
Nipah virus
Hendra virus
Hendra virus
Hendra virus
Hendra virus
Hendra virus
Hendra virus
Nipah virus
Material
Aluminum
Aluminum
Blood
Polystyrene
Polystyrene
Polystyrene
Polystyrene
Mango flesh
Mango flesh
Mango flesh
Mango flesh
Mango flesh
Mango flesh
Mango flesh
Environmental Condition
20°C
20°C
Dried
Desiccated, 22°C
Desiccated, 37°C
Desiccated, 22°C
Desiccated, 37°C
22°C, pH3.5
22°C, pH 4.5
22°C, pH 5
37°C, pH3.5
37°C, pH4.5
37°C, pH 5
22°C, pH3.5
Shortest Duration
Longest Duration Pi
without Study
with Persistence
Persistence
2.91hourst Sinclair et al.
(T99 value) (2008)
2.16 hourst
(T99 value)
5 dayst
30 minutes 1 hour Fogartyetal.
5 minutes 15 minutes (2008)
Ihour*
5 minutes 15 minutes
18 minutes
(half-life)
3.5 hours
(half-life)
22.4 hours
(half-life)
24 minutes
(half-life)
30 minutes
(half-life)
5.9 hours
(half-life)
1.4 hours
(half-life)
38
-------
Virus
Nipah virus
Nipah virus
Nipah virus
Nipah virus
Nipah virus
Ebola virus
Ebola virus
Ebola virus
Ebola virus
Ebola virus
Ebola virus
Ebola virus
Marburg virus
Marburg virus
Material
Mango flesh
Mango flesh
Mango flesh
Mango flesh
Mango flesh
Polyvinyl chloride
Polyvinyl chloride
Glass
Glass
Glass
Stainless steel
Stainless steel
Polyvinyl chloride
Polyvinyl chloride
Environmental Condition
22°C, PH4.5
22°C, pH 5
37°C, PH3.5
37°C, PH4.5
37"C, pH 5
4°C, 55% RH, applied in guinea pig serum
and tissue culture medium
Room temperature, 55% RH, applied in
guinea pig serum and tissue culture medium
4°C, 55% RH, applied in guinea pig serum
4°C, 55% RH, applied in tissue culture
medium
Room temperature, 55% RH, applied in
guinea pig serum and tissue culture medium
4°C, 55% RH, applied in guinea pig serum
and tissue culture medium
Room temperature, 55% RH, applied in
guinea pig serum and tissue culture medium
4°C, 55% RH, applied in guinea pig serum
4°C, 55% RH, applied in tissue culture
medium
Longest Duration
with Persistence
5.5 hours
(half-life)
30.3 hours
(half-life)
12 minutes
(half-life)
30 minutes
(half-life)
2.2 hours
(half-life)
14 days
<2 days
14 days
50 days*
<2 days
<2 days
<2 days
14 days
26 days
Shortest Duration
without Study
Persistence
-
-
-
-
Piercyetal. (2010)
26 days
2 days
26 days
-
2 days
2 days
2 days
26 days
50 days
39
-------
Virus Material
Marburg virus Polyvinyl chloride
Marburg virus Glass
Marburg virus Glass
Marburg virus Glass
Marburg virus Stainless steel
Marburg virus Stainless steel
VEE virus Rubber
Painted
VEE virus
aluminum
VEE virus Glass
Lassa virus Glass
Ebola virus Glass
Rift Valley fever Blood spotted on
virus filter paper
Environmental Condition
Room temperature, 55% RH, applied in
guinea pig serum and tissue culture medium
4°C, 55% RH, applied in guinea pig serum
4°C, 55% RH, applied in tissue culture
medium
Room temperature, 55% RH, applied in
guinea pig serum and tissue culture medium
4°C, 55% RH, applied in guinea pig serum
and tissue culture medium
Room temperature, 55% RH, applied in
guinea pig serum and tissue culture medium
20°C to 25°C, 30% to 40% RH
20°C to 25°C, 30% to 40% RH
20°C to 25°C, 30% to 40% RH
20°C to 25°C, 30% to 40% RH
20°C to 25°C, 30% to 40% RH
Stored dry at room temperature
-- Not tested/not reported.
* The longest duration tested (i.e., the actual persistence duration could be longer).
t Data obtained from a literature review, the shortest duration without persistence was not
t The longest duration with persistence was based on the time needed to decrease the viral
Shortest Duration
Longest Duration Pi
without Study
with Persistence _ . „
Persistence
<2 days 2 days
14 days 26 days
26 days 50 days
<2 days 2 days
<2 days 2 days
<2 days 2 days
7 days* Sagripanti et al.
(2010)
7 days*
11.4 days*
9.7 days*
5.9 days*
Naslund et al.
2 days 3 days (2(m)
reported.
load by 4 logio.
40
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Piercy et al. (2010) studied the persistence of Ebola and Marburg viruses on plastic (polyvinyl chloride),
stainless steel, and glass. The viruses were applied in liquid suspensions (tissue culture medium or
guinea pig serum) and allowed to dry for 30 minutes. The inoculated materials were held at 4°C or room
temperature at 55% RH. Neither virus was recovered from any surface held at room temperature nor
from stainless steel at 4°C (the shortest duration tested in each instance was 2 days). At 4°C, the Ebola
and Marburg viruses survived more than 14 days on plastic and glass. In fact, Marburg virus survived 26
days on glass and plastic, and Ebola virus persisted 50 days on glass (the longest duration tested) (Piercy
etal., 2010).
Sagripanti et al. (2010) deposited 3 x 10s plaque forming units (PFU) of VEE virus on glass, rubber, and
painted aluminum and held the inoculated materials in the dark at 20°C to 25°C and 30% to 40% RH. The
virus survived for 7 days (the longest duration tested) on each material. Sagripanti et al. (2010)
conducted similar testing with VEE virus, Lassa virus, and Ebola virus inoculated onto glass (held at 20°C
to 25°C, 30% to 40% RH, and shielded from light). The time needed to decrease the viral load by 4 logw
(T4) was 11.4 days for VEE virus, 9.7 days for Lassa virus, and 5.9 days for Ebola virus (Sagripanti et al.,
2010).
Naslund et al. (2011) spotted blood (containing 4,800 or 120,000 PFU of Rift Valley fever virus) onto
Nobuto filter papers. The filter papers were stored at room temperature. At various time points the
filter papers were eluted with RNase-free water and the supernatants were added to Vero cells. The
Vero cells were infected from extracts of the filter papers inoculated with 120,000 PFU of Rift Valley
fever virus after 2 days of storage but not after 3 days. Testing associated with Rift Valley fever virus at
4,800 PFU also demonstrated infected Vero cells (i.e., virus persistence) after 1 day of storage but not
after 2 days (Naslund et al., 2011).
10.3. Viral Encephalitis and Hemorrhagic Fever Agents in Water
Sinclair et al. (2008) estimated T99 values of 96 hours (4 days) for the vaccine strain of the yellow fever
virus (a hemorrhagic fever agent), T99 values of 165 to 710 hours (29.6 days) for the adenovirus, which
can cause encephalitis, and T99 values of 30 hours to 69.4 days for the hantavirus, which can cause
hemorrhagic fever. For hantavirus, the longest T99 values (69.4, 50, and 31.4 days) were associated with
tests conducted at 4°C; the next highest T99 value was 13 days at 23°C (based on studies from Hardestam
et al., 2007 and Kallio et al., 2006). Sinclair et al. (2008) also provided data for Sicilian virus that indicated
an even longer T99 value of 325 days at 4°C (based on research from Hardestam et al., 2007). Since the
Sicilian virus is known to induce fever but not encephalitis or hemorrhagic fever, these data are not
further included in this report. Two additional studies were identified that assessed virus persistence in
water as summarized in the following paragraphs.
Fitzgibbon and Sagripanti (2008) tested the persistence of VEE virus at approximately 2.5 x 10s PFU ml"1
in distilled-deionized (unchlorinated) water and tap water with a chlorine level of 4 to 5 parts per million
(ppm). Testing was conducted at 21°C, and the distilled-deionized water was also tested at 4°C and 30°C.
VEE virus persisted 21 days (the longest duration tested) in distilled-deionized water held at 4°C, 21°C,
41
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and 30°C; log reductions in virus titer were 0.18 at 4°C, 2.59 at 21°C, and 2.94 at 30°C. In tap water at
21°C, VEE virus persisted for 17 days but became non-detectable within 21 days (Fitzgibbon and
Sagripanti, 2008).
Wade et al. (2010) found that VEE virus at 1 x 10s PFU ml"1 was inactivated within 1 hour in tap water at
21°C with 1 mg L"1 free available chlorine or 2 mg L"1 total bromine. In the absence of the chlorine and
bromine, the VEE virus remained infective in tap water for 4 hours (the longest duration tested) with
little reduction in the initial titer level (Wade et al., 2010). The available persistence data identified for
viral encephalitis and hemorrhagic fever agents in water is also summarized in Table 14.
Table 14. Viral Encephalitis and Hemorrhagic Fever Agents Persistence in Water
Virus
Hantavirus
Adenovirus
Yellow fever
virus
Environmental
Condition
4°C
6°C
37°C
Longest Duration Shortest Duration
with Persistence without Persistence
69.4 dayst
(T99 value)
29.6 dayst
(T99 value)
4 dayst
(T99 value)
Study
Sinclair etal. (2008)
VEE virus
VEE virus
VEE virus
VEE virus
Distilled-deionized
(unchlorinated)
water, 4°C, 21°C,
and30°C
Tap water (4 to 5
ppm chlorine),
21°C
Tap water (1 mg L"
^ree available
chlorine or 2 mg L"
1 total bromine),
21°C
Tap water
(without chlorine
or bromine), 21°C
21 days*
Fitzgibbon and
Sagripanti (2008)
17 days
<60 minutes
4 hours*
21 days
60 minutes
Wade etal. (2010)
— Not tested/not reported.
* The longest duration tested (i.e., the actual persistence duration could be longer).
t Data obtained from a literature review, the shortest duration without persistence was not reported.
42
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10.4. Viral Encephalitis and Hemorrhagic Fever Agents Persistence Gaps
No data were identified with regard to the persistence of viral encephalitis or hemorrhagic fever agents
in soil. Persistence data were only identified for viral hemorrhagic fever agents in aerosols and on
fomites. The new aerosol persistence data for the Ebola and Marburg viruses, identified since Sinclair et
al. (2008), was limited to testing at one environmental condition (approximately 50% RH and 22°C;
Piercy et al., 2010; Smither et al., 2011). Piercy et al. (2010) noted that the assessment of virus
persistence in aerosols is affected by RH, the suspension and approach used to aerosolize the virus, and
methods used to enumerate the virus. Although all tested viruses persisted 90 minutes in aerosols,
Piercy et al. (2010) reported differences in decay rates by virus. Zaire Ebola virus and Lake Victoria
Marburg virus had similar decay rates, but the decay rate for the Reston Ebola virus was significantly
lower than for the Zaire Ebola virus and Lake Victoria Marburg virus (Piercy et al., 2010). On fomites,
Ebola and Marburg viruses were affected by material type (less persistent on stainless steel than plastic
and glass) and temperature (less persistent at room temperature than at 4°C) (Piercy et al., 2010). None
of the studies identified for viral encephalitis and hemorrhagic fever agents assessed the impact of
sunlight on persistence. Persistence data in water (since Sinclair et al., 2008) were limited to research
with VEE virus.
11. Yersinia pestis Persistence
Plague in humans typically results from Y. pestis infected fleas transmitting the bacterium from rodents
to humans. Human plague is generally associated with geographic areas having Y. pestis circulating in
the rodent population. Soil may serve as a reservoir for Y. pestis, which might explain prolonged periods
where the bacterium is not found in host or vector populations (Drancourt et al., 2006). The persistence
of Y. pestis in the environment (e.g., soil) might lead to animals becoming infected (e.g., burrowing
rodents), which could lead to a new transmission cycle in rodents and fleas (Drancourt et al., 2006). As
reported by Pawlowski et al. (2011a), "a growing body of evidence suggests that Y. pestis can survive
without a host for extended periods under certain environmental conditions while, in many cases,
retaining infectivity."
11.1. Y. pestis in Aerosols
Sinclair et al. (2008) reviewed one study (Won and Ross, 1966) investigating the persistence of Y. pestis
as an aerosol. The estimated T99 values were less than 1 hour at 26°C, including 34 minutes at 87% RH,
57 minutes at 50% RH, and 45 minutes at 20% RH. The lowest T99 value occurred at 87% RH, and Sinclair
et al. (2008) noted that organism survival decreased rapidly as RH increased above 50%. Won and Ross
(1966) used both 1% peptone and heart infusion broth as the diluents in the assay. At 20% to 50% RH, Y.
pestis had exponential decay with both diluents. However, at 65% and 87% RH viability of the
aerosolized Y. pestis was adversely affected with 1% peptone (no viability detected after 20 minutes),
43
-------
but viability was detected after 90 minutes using heart infusion broth. No more recent data of the
persistence of Y. pestis in aerosols were identified with this review.
11.2. Y. pestis on Fomites
Sinclair et al. (2008) reviewed two studies (Wilkinson, 1966; Rose et al., 2003) investigating the
persistence of Y. pestis on fomites. The estimated T99 values ranged from 7 minutes (on stainless steel at
30°C and 52% RH) to 47.2 hours (on paper at 18°Cto 22°C and 55% RH). Most of the testing was
conducted at 18°Cto 22°Cand 55% RH (Roseetal., 2003), and under these conditions /. pestis survived
longer (higher T99 values) on paper than stainless steel, polyethylene, or glass (Sinclair et al., 2008).
Persistence testing was conducted with Y. pestis following inoculation onto aluminum, carpet, keyboard
keys, and painted joint tape (Ryan, 2010). Material coupons were inoculated with 2.9 x 107 viable Y.
pestis in 100 ul aliquots of stock suspension (trypticase soy broth). The inoculated coupons were held at
approximately 50% RH and 20°C. At various time points, the coupons were placed in PBS and agitated on
an orbital shaker to extract the Y. pestis from the materials. Aliquots of the undiluted extract and
associated serial dilutions were spread plated onto tryptic soy agar and incubated for up to 72 hours at
37°C. Y. pestis was found to persist (although the mean recovery was <1%) on aluminum and painted
joint tape for 7 days (the longest duration tested).The longest Y. pestis persistence (also <1% mean
recoveries) on the other materials was 3 days on the keyboard keys and 8 hours on carpet.
11.3. Y. pestis in Soil
Three sources reviewed in Sinclair et al. (2008) (Breneva et al., 2005; Breneva et al., 2006; Mitscherlich
and Marth, 1984) reported that Y. pestis persisted for 3.5 months in soil at room temperature and
persisted for >10 months in soil at colder temperatures (i.e., 4°C to 8°C). Drancourt et al. (2006)
demonstrated the persistence of Y. pestis in autoclaved, hydrated sand for at least 6 months. (Additional
details describing the test conditions were not provided by Drancourt et al. [2006].) In a natural setting,
Eisen et al. (2008) found that Y. pestis could persist at least 24 days in Arizona soil during late October in
an area with limited exposure to sunlight (the intensity/duration of exposure to sunlight was not
reported).
Ayyadurai et al. (2008) inoculated sterile soil with /. pestis at 10s CPU g"1, which was held at 18.7°Cto
24°C (not exposed to sunlight). Y. pestis was able to be identified from soil cultures for 30 weeks, but
thereafter contamination with Pseudomonas species of bacteria interfered with the culturing of Y.
pestis. However, Y. pestis was found to be viable and virulent for up to 40 weeks (the longest duration
tested) based on inoculations of soil extracts into mice. The mice died within 72 hours post-infection and
the blood cultures were positive for Y. pestis (Ayyadurai et al., 2008).
A review article by Eisen and Gage (2009) noted that several studies have shown persistence of Y. pestis
in soil. Many of the specific details of these tests were not provided (exact durations of persistence and
44
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specific environmental conditions tested), and many of the source documents were not in English. Eisen
and Gage (2009) also noted that other researchers have suggested that Y. pestis survives in soil inside
protozoa, within a biofilm, or as a latent form, rather than as a free-living, metabolically active
bacterium. Persistence data for Y. pestis in soil is summarized in Table 15.
Table 15. Y. pestis Persistence in Soil
Y. pestis
Isolate
—
Isolate
(unspecified)
from soil in
Arizona
6/69M
Environmental
Condition
4°Cto8°C
Room
temperature
Autoclaved,
hydrated sand
Arizona soil during
late October in an
area with limited
exposure to
sunlight (UV light)
18.7°Cto24°C
Longest Duration Shortest Duration
with Persistence without Persistence
10 months*
3.5 monthst
6 months*
24 days*
40 weeks*
Study
Sinclair etal. (2008)
Drancourt et al.
(2006)
Eisen et al. (2008)
Ayyadurai et al.
(2008)
-- Not tested/not reported.
* The longest duration tested (i.e., the actual persistence duration could be longer).
t Data obtained from a literature review, the shortest duration without persistence was not reported.
11.4. Y. pestis in Water
From the Sinclair et al. (2008) review of Mitscherlich and Marth (1984), Y. pestis is able to survive in tap
and well water for 16 days. Sinclair et al. (2008) also reported that Y. pestis may enter into a VBNC state
in water as well as possibly being able to survive inside amoeba. Four additional studies on the
persistence of Y. pestis in water were identified that are summarized in the following paragraphs.
Pawlowski et al. (2011a) inoculated Y. pestis (at approximately 10s to 107 CFU ml"1) into various types of
water held at 4°C. The bacterium remained culturable for 28 days (the longest duration tested) in
sterilized river water with nearly 107 CFU ml"1 recovered, artificial sea water with 10s CFU ml"1
recovered, and non-autoclaved tap water with approximately 101 CFU ml"1 recovered. Y. pestis
remained culturable for only 14 to 21 days in autoclaved tap water, and Pawlowski et al. (2011a)
confirmed that the bacterium had entered a VBNC state.
Pawlowski et al. (2011b) inoculated Y. pestis at 5 x 10s CFU ml"1 into filtered or autoclaved river water. Y.
pestis was culturable from the autoclaved river water for >3 years, while the bacterium persisted for 200
days in the filtered river water it was no longer culturable within 265 days in the filtered river water.
Pawlowski et al. (2011b) noted that the bacterium Hylemonella gracilis actually dominated the filtered
river water, apparently inhibiting the long-term survival of Y. pestis.
45
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Torosian et al. (2009) evaluated the persistence of several strains of Y. pestis in sterilized bottled spring
water held at 26°C. Approximately 2 x 104 CPU ml"1 of Y. pestis was inoculated into the water and the
bacteria remained culturable for at least 74 days with some strains persisting for at least 2 years (the
longest duration tested). The persistence testing was initiated with seed stock generated directly from
frozen stock culture or from agar slants. More specifically, the following Y. pestis strains (generated from
frozen culture): CO92, ZE94, EV76 Bru, JBH, UNH IB, KIM 10, O19 Tn5, CSH23, K25 Icr, and O19 Ca'6 had
persistent durations of 94 to 683 days. Other Y. pestis strains (UNH 1A, K25 pgm, K25 pst, 5.5, EV76 51F
RP, and A1122) generated from frozen culture had persistent durations of at least 2 years. The following
Y. pestis strains (generated from agar slants) persisted for 74 to 221 days: Harbin, Nepal, UNH 1A, UNH
IB, ZE94, CO92, PB6, PB6 DP, Pexu, K25 Icr, O19 Ca'6, and K25 pst. Torosian et al. (2009) noted that
unlike strains originating from frozen stock, none of the strains started from agar slants persisted for 2
years. The authors discussed the possibility that the frozen cells expressed an adaptive tolerance that
may have affected persistence. The source of the seed stock used by Torosian et al. (2009) is noted
within the "/. pestis strain" column of Table 16. None of the other /. pestis persistence studies in water
reported using cultures initiated directly from the frozen state.
Gilbert and Rose (2012) used autoclaved, dechlorinated municipal water inoculated with 10s CPU ml"1 Y.
pestis to assess persistence at 5°C and 25°C. Y. pestis was culturable for 14 days when held at 25°C and
culturable for 1 day when held at 5°C (Table 16). In addition to culturing, viability was assessed by
measuring metabolic (i.e., esterase) activity. Based on esterase production, Y. pestis remained viable for
30 days (the longest study duration) at 5°C and 25°C, suggesting that the bacterium entered a VBNC
state (persistence based on metabolic activity is not presented in Table 16). Data on the persistence of /.
pestis in water (i.e., via culturability) are summarized in Table 16.
46
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Table 16. F. pestis Persistence in Water
Y. pestis Strain
Harbin 35
Harbin 35
Harbin 35
Harbin 35
A1122
A1122
CO92, ZE94, EV76 Bru,
JBH, UNH IB, KIM 10,
019 Tn5, CSH23, K25 Icr,
inr\ O1 Q P^»~6 lnanam+ar\
Environmental
Condition
Tap water and well
water
Sterilized (filtered and
autoclaved) river
water, 4°C
Artificial sea water,
4°C
Non-autoclaved tap
water, 4°C
Autoclaved tap water,
4°C
Filtered river water
Autoclaved river
water
Sterilized bottled
spring water, 26°C
Longest Duration
with Persistence
16 dayst
28 days*
28 days*
28 days*
14 days
200 days
3 years*
94 days to 683
days
Shortest Duration
without Study
Persistence
Sinclair et al.
(2008)
Pawlowski et
al. (2011a)
-
-
21 days
265 days Pawlowski et
al. (2011b)
Torosian et al.
(2009)
from frozen culture)
UNH 1A, K25 pgm, K25
pst, 5.5, EV76 51F RP,
and A1122 (generated
from frozen culture)
Harbin, Nepal, UNH 1A,
UNH IB, ZE94, CO92,
PB6, PB6 DP, Pexu, K25
Icr, O19 Ca'6, and K25
pst (generated from
agar slants)
A1122 and AZ 94-0666
A1122 and AZ 94-0666
Sterilized bottled
spring water, 26°C
Sterilized bottled
spring water, 26°C
Autoclaved,
dechlorinated
municipal water, 5°C
Autoclaved,
dechlorinated
municipal water, 25°C
2 years*
74 days to 221
days
Iday
14 days
2 days
21 days
Gilbert and
Rose(2012)
-- Not tested/not reported.
* The longest duration tested (i.e., the actual persistence duration could be longer).
t Data obtained from a literature review, the shortest duration without persistence was not reported.
47
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11.5. Y. pestis Persistence Gaps
Relatively little data were identified on the persistence of Y. pestis in aerosols and fomites, and few
studies were found focusing on the impact of various environmental parameters (e.g., humidity,
sunlight, and temperature) on persistence. Most persistence data on Y. pestis were associated with
survivability in water, including testing at different temperatures. Interestingly, Y. pestis in water ranged
from days to years with trends between environmental condition and persistence not apparent. Gilbert
and Rose (2012) noted that some of the variability associated with Y. pestis persistence in water might
be attributed to differences in water chemistry, organism strain or growth phase, growth media for
culturing and recovery, and inoculum levels.
12. Summary
The range of persistence durations (shortest and longest) identified by this review are summarized by
agent and medium in Table 17. The table also includes the environmental condition associated with
each value and identifies agent/medium combinations lacking persistence data. For agents with
persistence data available on fomites, soil, or water, there are environmental conditions conducive to
persistence for multiple days. In fact, several agent/medium combinations are associated with
persistence of a month (or more) in duration including Brucella species on fomites, soil, and water; 6.
mallei on fomites and water; B. pseudomallei in soil and water; F. tularensis in water; viral encephalitis
and hemorrhagic fever agents on fomites and water; and Y. pestis in soil and water. Although C. burnetii
can develop a resistant spore-like form, very limited environmental persistence data was identified.
Persistence data were less readily available, and much of the aerosol data summarized in Table 17 were
obtained from Sinclair et al. (2008).
Factors that affect persistence are discussed in the following paragraphs by medium (and agent) as
available. Generally, persistence may be affected by the various environmental media and factors (e.g.,
temperature, RH, and sunlight), subspecies and strains, preparation and application methods, and
nutrient conditions. Persistence may be increased by the presence of organisms that serve as hosts or to
which the biological agents have symbiotic relationships (e.g., possibly amoeba, earthworms), or
persistence may decrease based on the present of competing and/or predatory organisms in the
environment. Analytical methods may also affect comparison or interpretation of persistence results
across studies (e.g., different culture media, incubation temperatures and times may be employed).
Similarly, the estimates of persistence may differ depending on the technique used (molecular,
culturing, counting, etc.) and the relative occurrence of VBNC bacteria associated with the various
techniques.
Aerosol
Persistence data in aerosols were lacking for B. anthracis, Brucella species, B. mallei, B. pseudomallei,
and C. burnetii. Persistence research was conducted with Ebola and Marburg viruses in aerosols,
although only one environmental condition (50-55% RH and 22°C) was tested. Other viral encephalitis or
hemorrhagic fever agents may behave differently than the viruses tested, especially under different
48
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environmental conditions. As reported by Sinclair et al. (2008) some viruses (i.e., Japanese encephalitis
virus and VEE virus) appeared to have an inverse relationship with RH. For Y. pestis, Sinclair et al. (2008)
noted that in aerosols the bacterium persistence decreased rapidly as RH increased above 50%,
although the decrease was much less rapid when heart infusion broth was used as the diluent rather
than 1% peptone (Won and Ross, 1966). Based on work with aerosolized F. tularensis, Hood (2009)
reported that unidentified constituents in outdoor air (possibly olefins) may reduce the persistence of
non-spore forming microorganisms.
Fomite
Some persistence data on fomites were available for all agents except C. burnetii. The persistence
statements identified for B. mallei on fomites were likely made based on observation in the field rather
than controlled laboratory studies. Nevertheless, it appears that 6. mallei persistence is greatest under
warm humid or moist conditions (Dvorak and Spickler, 2008).
49
-------
Table 17. Summary of Agent Persistence in the Environment
Agent Medium
Conditions with Shortest Duration Reported
Persistence
Duration
Associated Environmental
Condition
Reference
Conditions with Longest Duration Reported
Persistence
Duration
Associated Environmental
Condition
Reference
Aerosol
Fomite
Bacillus
anthracis*
Soil
Water
Aerosol
Fomite
Brucella species
(e.g., suis,
melitensis,
abortus, etc.) Soil
Water
Aerosol
Fomite
Burkholderia
mallei
Soil
Water
-
25°C, 80% RH on stainless
6 hours steel coated with silver and
zinc zeolite paint
—
3 days Distilled water
-
22"C, 45% RH on painted
4 hours
joint tape
<4 days Dried soil
<1 day 37°C
-
Environmental survival
(specific fomites not
3 weeks .
identified) in wet, humid, or
dark conditions
-
Dechlorinated municipal
(ay water, 5°C and 25°C
Galeano et
al. (2003) ? daVSt
Sinclair et
al. (2008) 6 daVS
Ryan
(2010) 56 daVSt
Nicoletti
(1980) 43 daVS
Nicoletti
(1980) 7? daVS
Dvorak and
Spickler 3 months
(2008)
Gilbert and
Rose (2012) 28 dayS
-
37°C on polystyrene and
glass as a biofilm in BHI
broth
—
Water
-
22°C, 40% RH on aluminum
and glass; and 5°C, 30% RH
on aluminum, glass, and
wood
Bison partition sites in
Greater Yellowstone,
identified in April
Room temperature
-
On stable bedding, troughs,
and harness equipment
-
Tap water, room
temperature
Lee et al.
(2007)
Sinclair et
al. (2008)
Calfee and
Wendling
(2012)
Aune et al.
(2012)
Nicoletti
(1980)
Malik etal.
(2012)
Miller et al.
(1948)
50
-------
Agent Medium
Conditions with Shortest Duration Reported
Persistence
Duration
Associated Environmental
Condition
Reference
Conditions with Longest Duration Reported
Persistence
Duration
Associated Environmental
Condition
Reference
Aerosol
Applied in Butterfield buffer
Fomite
Burkholderia
pseudomallei
Soil
Water
Aerosol
Fomite
Coxiella
burnetii Soil
Water
Aerosol
Francisella
tularensis
Fomite
Soil
Water
\.\J QICtJJf \JCt\J*^l f
6 hours polyethylene, and stainless
steel; and applied in BHI
broth to stainless steel
Soil inoculated with
<10 days antagonistic bacteria (e.g.,
6. multivorans)
60 minutes Water exposed to sunlight
-
20 dayst 20°C, 4°C, and -20°C
-
29 minutes
50% RH, wet dissemination
(T99 value)
20 minutes Desiccated on filter paper
-
Dechlorinated municipal
V water, 5°C and 25°C
Shams et al.
(2007) ayS
Lin et al
(2011)' 30months
Sagripanti et 16 yearst
al. (2009)
Evstigneeva
etal. (2007)
Sinclair et al.
(2008); Cox
(1971); Cox 5 dayS
and Goldberg
/ •! C\~i *") \
(1972)
\ /
Faith et al.
/->r,i->\ 16 dayst
(2012)
Gilbert and
Rose (2012) 34 dayst
/-VfJfJIICU III LJIII Ul *_/LI 1 H_/
paper, polyethylene, and
stainless steel
Soil stored in plastic bags at
ambient temperature (13°C
to33°C).
Distilled water, 25°C
-
20°C, 4°C, and -20°C
-
90% RH, wet dissemination
37°C, 0% RH on stainless
steel
-
Brackish-water, 21°C
Shams et al.
(2007)
Thomas and
Forbes-
Faulkner
/ *1 OO*1 \
(1981)
Pumpuang
etal. (2011)
Evstigneeva
etal. (2007)
Sinclair et
al. (2008);
Cox (1971);
Cox and
Goldberg
(1972)
Wilkinson
(1966)
Berrada and
Telford
/ *"> r\ *i *i \
(2011)
51
-------
Agent
Conditions with Shortest Duration Reported
Medium Persistence
Duration
Associated
Environmental
Condition
Conditions with Longest Duration Reported
Persistence
Reference
Duration
Associated Environmental
Condition
Reference
Viral
encephalitis
and
hemorrhagic
fever agents
Yersinia pestis
Aerosol
Fomite
Soil
Water
Aerosol
Fomite
Soil
Water
1 hourt
5 minutes
<60
minutes
34 minutes
(T99 value)
7 minutes
(T99 value)
24 dayst
Iday
22"C, 50% RH
Desiccated, 37°C
-
Tap water (1 mg L"1 free
available chlorine or
2 mg L1 total bromine), 21°C
26"C, 87% RH
30°C, 52% RH on metal
(stainless steel)
Arizona soil during late
October in an area with
limited exposure to UV light
Dechlorinated municipal
water, 5°C
Smither et al.
(2011)
Fogarty et al.
(2008)
Wade et al.
(2010)
Sinclair et al.
(2008)
Sinclair et al.
(2008);
Wilkinson
MQfifil
I j.J\J\J I
Eisen et al.
(2008)
Gilbert and
Rose (2012
120 days
(T99 value)
50 dayst
69 days
(T99 value)
57 minutes
(T99 value)
7 dayst
10 monthst
3 yearst
21°C, 23% RH
4°C, 55% RH in tissue
culture medium on glass
-
4°C
26°C, 50% RH
20°C, 50% RH on aluminum
and painted joint tape
4°Cto8°C
Autoclaved river water
Sinclair et
al. (2008)
Piercy et al.
(2010)
Sinclair et
al. (2008)
Sinclair et
al. (2008)
Ryan (2010)
Sinclair et
al. (2008)
Pawlowski
etal.
(2011b)
-- Not tested/not reported.
* This review focused on vegetative B. anthracis only.
t The longest duration tested (i.e., the actual persistence duration could be longer).
52
-------
One of the unique aspects of persistence testing with fomites is the influence of the material type,
which affects persistence and recovery of agent from the material. Most of the testing with fomites was
conducted with relatively smooth non-porous surfaces (e.g., aluminum, glass, keyboard keys, plastic,
polyvinyl chloride, stainless steel). Limited testing was conducted on concrete and wood (with B. suis)
and on carpet, painted joint tape, and filter paper (with F. tularensis). Differences between fomite
materials and persistence were noted for Y. pestis. For example, Y. pestis persisted longer on paper than
stainless steel, polyethylene, or glass (Sinclair et al., 2008). Y. pestis persistence was shortest on carpet,
when compared with aluminum, painted joint tape, and keyboard keys (Ryan, 2010).
Shams et al. (2007) found that the persistence durations for B. pseudomallei on fomites differed
depending upon the type of suspension used for application. Longer persistence was observed when 6.
pseudomallei was applied in BHI broth rather than Butterfield buffer. Comparisons between the F.
tularensis fomite persistence work by Wilkinson (1966) and Faith et al. (2012) also indicated that results
may be affected by inoculum levels and inoculation approaches.
The persistence work with B. anthracis on fomites was conducted at 80% RH for <24 hours to avoid
desiccation or within nutrient broth to access the bacteria's ability to form biofilms. The ability of
vegetative B. anthracis to persist of surfaces after drying is unknown. The influence of cold
temperatures is also unknown for B. anthracis on fomites as all tests were conducted at >25°C. The
persistence work with B. suis on fomites (e.g., Ryan, 2010; US EPA, 2010b) was one of the few agents
that was investigated on multiple materials, at a warm (22°C) and cool (5°C) temperatures, and with and
without simulated sunlight. Some materials (e.g., painted joint tape and concrete) appeared to
adversely affect survival, as did the warmer temperatures and simulated sunlight. For F. tularensis,
Wilkinson (1966) generally found better survival at 25°C than 37°C, and better survival under low RH (0-
10%) than elevated RH (55-100%). Ebola and Marburg viruses survived better on glass and plastic at 4°C
than at room temperature (Piercy et al., 2010). Similarly, henipaviruses persisted on mango flesh and
polystyrene longer at 22°C than 37°C (Fogarty et al., 2008).
Soil
Persistence data in soil were identified for Brucella species, B. pseudomallei, C. burnetii, and Y. pestis.
For Brucella species in soil, environmental conditions such as sunlight, elevated temperatures, and dry
soil adversely affected persistence (Nicoletti, 1980; Jones et al., 2010). B. pseudomallei is associated with
decaying organic matter and seems to survive well in moist subsurface soil at warm temperatures (e.g.,
24°C to 42°C), although dry conditions and colder temperatures can be tolerated (Tong et al., 1996;
Larsen et al., 2013; Chen et al., 2003). C. burnetii was found to survive 20 days (the longest duration
tested) in soil held at 20°C, 4°C, and -20°C (Evstigneeva et al., 2007).
The importance of the soil environment as a reservoir or ecological niche for several agents is being
investigated for several agents including B. anthracis, B. pseudomallei, C. burnetii, and Y. pestis. The
rhizosphere of grasses may be important for the environmental survival of B. anthracis vegetative cells
and spores (Saile and Koehler, 2006) and B. pseudomallei (Kaestli et al., 2012). Soil amoeba might
provide an intracellular niche for C. burnetii (La Scola and Raoult, 2001) and Y. pestis (Eisen and Gage,
2009). Bacteriophages and worms may also have an important role in the environmental survival of 6.
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anthracis vegetative cells within soil (Schuch and Fischetti, 2009). The potential association with 6.
anthracis and earthworms is interesting as both prefer alkaline soils with high calcium and organic
matter content (Hugh-Jones and Blackburn, 2009; Schuch et al., 2010). Some soil bacteria might inhibit
the growth of other agents. For example, B. multivorans has been shown to inhibit B. pseudomallei
growth (Lin et al., 2011). The interactions of a released agent with other microorganism might enhance
or inhibit the agent's survival and/or establishment of a viable population in the environment. Such
dynamics require additional research.
Water
Persistence in water may be affected by sunlight, temperature, nutrients, and salinity. Sunlight has been
demonstrated to inhibit the persistence of B. pseudomallei in water (Sagripanti et al., 2009). Gilbert and
Rose (2012) found that B. pseudomallei and Y. pestis were culturable longer at 25°C than at 5°C. Other
researchers have found that B. pseudomallei can survive a wide range of environmental conditions
(including temperature, pH, and salinity) in water (Robertson et al., 2010). Interestingly many of the
studies with B. pseudomallei were conducted with sterilized water samples. It is uncertain how 6.
pseudomallei would survive in natural water and the unknown interactions with other environmental
microorganisms. Based on the data presented by Nicoletti (1980), Brucella species persistence appears
to be shortened by elevated temperatures. For F. tularensis, Berrada and Telford (2011) indicated that
the salt and sulfur content of brackish water may enhance the bacterium's persistence.
The phenomena of bacteria entering a VBNC state was noted for several agents including Brucella
species, B. mallei, B. pseudomallei, F. tularensis, and Y. pestis (Gilbert and Rose, 2012). The importance
of VBNC agents is an area in need of additional research. In addition, some agents appear to survive and
benefit from being ingested by amoeba, including B. anthracis (Dey et al., 2012), B. pseudomallei
(Sagripanti et al., 2009), F. tularensis (EI-Etr et al., 2009). Some bacteria including F. tularensis (Mahajan
et al., 2011)also have the ability to form biofilms that may be important for survival in water.
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