technical   BRIEF
             Persistence of Categories A and B Select Agents in
                                Environmental  Matrices

           Persistence is the ability of an organism to remain viable
           over time under a given environmental condition. For
           bacteria, viability is the ability to remain alive, whereas
           for viruses, it is the ability to remain infectious.
           Understanding a biological agent's ability to persist in the
           environment would help authorities to properly assess
           and respond in the event of the agent's release.

           The purpose of this brief is to summarize persistence
           data documented in an EPA literature review (U.S. EPA,
           2014a) entitled Persistence of Categories A and B Select
           Agents in Environmental Matrices (EPA/600/R-14/0741
           Category A biological agents cause high rates of
           mortality and are easily disseminated or easily
           transmitted from person to person. Category B agents
           cause illness with low mortality and are moderately easy
           to disseminate. Assessment of persistence data is
           important to (1) allow informed emergency response and
           remediation decisions following a contamination incident,
           and (2) identify gaps in the current state of the science
           so research can be focused toward closing these gaps.

           This brief provides a short summary of the open
           literature findings published to date, specifically for
           biological agent persistence.  These data cover
           numerous agents, as well as various environmental
           conditions and surface types.  This brief reports the
           longest duration of persistence, which is from the time of
           inoculation to the last sampling interval that produced
           viable organisms. Several studies, however,  did not
           continue until organism could no longer be cultured; in
           those cases, the pathogen's actual duration of
           persistence could not be determined.
"Biological agent means any
microorganism (including, but
not limited to, bacteria,
viruses, fungi, rickettsiae, or
protozoa), or infectious
substance, or any naturally
occurring, bioengineered, or
synthesized component of
any such microorganism or
infectious substance, capable
of causing death, disease, or
other biological malfunction in
a human, an animal, a plant,
or another living organism;
deterioration of food, water,
equipment, supplies, or
material of any kind; or
deleterious alteration of the

42 Code of Federal
Regulations Part 73
 August 2015

 The literature review documents the duration of persistence of biological agents and provides
 information on environmental conditions and species interactions that either favor or hinder
 persistence. The environmental media considered were aerosol, soil, water, orfomite (an
 object, surface, or substance that can transmit pathogens). The review also identified the gaps
 in the scientific literature that impede a thorough understanding of the persistence of these
 pathogens.  This summary, based on the literature review, describes what is known and what
 is unknown about these conditions and interactions.

 As noted below, some species enter into a state where they are metabolically active, yet not
 culturable, with unknown viability. Ducret et al., 2014, concluded that the physiological
 significance of the state is unknown and controversial; it could be an adaptive state favoring
 long-term survival, it could be a deteriorating state leading to death, or it could be an injured
 state that prevents organisms from being cultured. The ability of an agent to subsequently
 cause infection or induce human disease following release into the environment was beyond
 the scope of the review. The importance of this metabolic state is an area in need of additional
 Bacillus anthracis

 B. anthracis is a spore-forming bacterial Category-A select agent, the causative agent of the
 disease anthrax. Under appropriate conditions, B. anthracis can form protective biofilms on
 glass and polyvinyl surfaces covered with nutrient media. B. anthracis strains involved in gut
 colonization have also formed biofilms. However, conditions and the overall persistence of the
 formed biofilms have yet to be explored.

 B. anthracis spores  can persist in soil for many years. Many symbiotic relationships between
 Bacillus species and soil-borne organisms have been reported in the literature. B. anthracis
 has been found to geminate in amoeba, and to colonize the hindgut of earthworms. There is
 some evidence that the interaction between earthworms and B. anthracis might be dependent
 on the presence of bacteriophages. It is unknown whether the colonies formed in the hindgut
 of earthworms and the  spores that germinate in amoeba can result in increased numbers of
 spores in the environment or if these spores would be virulent.

 The B. anthracis lifecycle and the earthworm lifecycle in the soil have similarities. Both prefer
 alkaline,  calcium rich, organic soils. In addition, anthrax outbreaks often occur following
 flooding, which is also when earthworms tend to migrate to the surface. The post-flooding
 anthrax outbreaks could be due to B. anthracis spores being carried upward to the soil surface
 and onto vegetation via colonized earthworm digestive systems. (For more information on the
 persistence  of B. anthracis spores in soil, see U.S. EPA, 2014b, entitled Literature  Review on
 Mechanisms that Affect Persistence of Bacillus anthracis in Soils.)

The literature search included persistence on surfaces, but little data on persistence on
surfaces was found.  Testing on fomites was not conducted at environmental conditions
<25C, <80% RH, or with simulated sunlight.

Virulent B. anthracis contains two plasmids, pX01 and pX02.  The pX01 plasmid has been
found to be required for spore germination within the amoeba. For strains lacking pX01,
bacteriophage infection of B. anthracis could restore functional gene activity necessary to
survive and replicate in earthworms, rhizosphere, biofilms, and soil. Researchers have
hypothesized that pX02 is lacking in multiple natural B. anthracis strains because it is not
required for proliferation. However, there is little information in the literature on conditions that
promote or inhibit proliferation.

Some studies have found that spores can germinate and propagate in the rhizosphere of a
common pasture grass, but do not germinate without the grass. Conflicting studies suggest
that presence of grass does not increase survival or multiplication of B. anthracis in soil, but
serves to promote transmission of B. anthracis to grazing hosts by attracting more hosts to the
area. Further research  is needed to determine if B. anthracis can take up the pX02 plasmid in
the environment and thus regain virulence.

Brucella species

Brucella species are  non-spore-forming bacterial Category-B  select agents, which cause the
disease brucellosis. Brucella species can enter a state where they are metabolically active, yet
not culturable, with unknown viability. Brucella melitensis is typically associated with goats,
Brucella suis is associated with swine, and Brucella abortus is often associated with cattle. All
three species can infect humans.

The persistence of B. melitensis and B. suis in water (<9 days at 5C and 25C) was
considerably lower than the persistence of B. abortus in water (>57 days at 8C and 20C). 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 form biofilms, which can enhance the bacteria's tolerance to desiccation. 6.
abortus appeared to survive better in moist soil than dry soil (66 days in wet soil compared to
<4 days in dried soil).

Brucella species can survive for several weeks in water, but the bacteria are sensitive to  heat,
with < I day survival at 37 C in lake water.  Brucella species also appear to be adversely
affected by exposure to sunlight. No data on the survival of Brucella species in aerosols was
found during this review. Research is needed on the mechanisms that contribute to the
environmental persistence of Brucella species including species-specific data.

Burkholderia mallei

B. mallei is a non-spore-forming non-motile bacterial category-B select agent, which causes
the disease glanders.  B. mallei can enter a state where they are metabolically active, yet not
cultivable, with unknown viability. B. mallei is primarily associated with horses and is not
expected to survive outside a host for long durations. However, B. 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. Although
three persistence studies were identified with B. mallei in water, the results were somewhat

No data on the survival of B. mallei in aerosols or on soil was found during this review.
Information on B. mallei persistence on fomites found during this review was not supported
with specific data or laboratory controlled studies.

Burkholderia pseudomallei

B. pseudomallei is a non-spore-forming motile bacterial category-B select agent, which causes
the disease melioidosis. B. pseudomallei can enter a state where they are metabolically active,
yet not culturable, with unknown viability. B. pseudomallei is associated with water and with
soil. B. pseudomallei occurs in tropical and sub-tropical climates and is associated with
decaying organic matter in the environment. B. pseudomallei can grow in anoxic (oxygen
deficient) environments.

Interactions with other organisms can influence persistence. B. pseudomallei is associated
with the rhizosphere, roots, and above ground parts of various grasses, especially non-native
grasses introduced for grazing animals. The relationship between B. pseudomallei and
vegetation is not well understood. Other soil bacteria  (e.g., Burkholderia multivorans) could
inhibit B. pseudomallei growth. On the other hand, B. pseudomallei can form biofilms and
survive in amoeba cysts and fungi.

Survival in various media is not well understood.  B. pseudomallei can be transported by
aerosols and has been isolated from  aerator spray associated  with a water treatment plant.
However, no data on the survival durations of B. pseudomallei in aerosols was found during
this review. Only one study investigated the persistence of B. pseudomallei on fomites.
However, the influence of different environmental conditions (temperature and humidity) on
survival on fomites  was not investigated.  Environmental factors dictating the occurrence of 6.
pseudomallei in soil and water are not well established.

Similarly, survival in soil and the effect of sunlight is not well understood. Subsurface samples
(e.g.,  at a 25-60 centimeter [cm] depth) are more likely to yield B. pseudomallei than surface
soil. B. pseudomallei is infrequently found in surface soil, possibly because the organism
might be adversely affected by sunlight. However, the review found no definitive studies on the
effects of sunlight on B. pseudomallei persistence. It is known that B. pseudomallei were killed

by radiation from an UV lamp (465 microwatt [|jW]/square centimeter [cm2] for 7.75 minutes),
but radiation associated with natural sunlight can be absorbed by other materials thereby
limiting sunlight's killing effect.

Other factors also affect persistence in soils. Optimal temperatures for B. pseudomallei
growth in soil are 37C to 42C, although some strains were able to grow at 4C. Optimal soil
pH was 6.5 to 7.5.   B. pseudomallei appears to benefit from moist soil, but can persist in  soil
that gradually dries. B. pseudomallei was not recovered following inoculation into dry soil, but
was recovered from dry soil in farmed areas suggesting that factors other than moisture are
needed to support B. pseudomallei growth. Under conditions of low water content, organic
matter in the soil might enable B. pseudomallei to survive.

Coxiella burnetii

C. burnetii is an obligate intracellular bacterial category-B select agent, which causes Q fever.
Commonly, barnyard dust from infected cattle, sheep, and goats transmits the disease to
humans.  C. burnetii can be aerosolized and transported by wind long distances. C. burnetii is
not known to be strongly affected by high or low temperatures, drought, or humidity levels. No
data on the persistence of C. burnetii in water was found during this review.

There are a number of environmental  reservoirs that could aid in the persistence and
transmission  of C. burnetii. C.  burnetii can invade a variety of hosts including amoebae, ticks,
birds, and mammals. It appears likely  that these hosts facilitate the dissemination of the
bacterium throughout the environment. C. burnetii was able to survive within amoeba for 6
weeks; soil amoeba could provide an intracellular niche for the survival of C. burnetii in a
spore-like form. Epidemiological evidence suggests  that C. burnetii may be carried over
considerable distances on fomites.

There are uncertainties and unknowns in the information on persistence for C. burnetii. 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.  Persistence might vary by the form of C. burnetii excreted from the host, but the specific
forms excreted in milk, feces or placentas are unknown.  In addition, much of the persistence
data of C. burnetii (in aerosol and fomites studies, and in some soil studies) were based on
environmental sampling associated with Q fever outbreaks and viability was not assessed.
Also, the studies were not conducted in controlled laboratory settings, so
recontamination/cross-contamination could have occurred.

Francisella tularensis

F. tularensis is a non-spore-forming bacterial category-A select agent, which causes tularemia,
also called rabbit fever. F. tularensis can enter into a state where they are metabolically active,
yet not culturable, with unknown viability. F. tularensis can infect mammals, arthropods, and

protozoans. Virulent strains of F. tularensis were shown to survive in the cysts of
Acanthamoeba castellanii for at least 3 weeks post-infection.

F. tularensis can survive in diverse environments, but the mechanism by which F. tularensis
persists or establishes an environmental reservoir following a release is unknown. 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. F. tularensis
survived longer in brackish water than fresh water. The salt and sulfur content of the brackish
water may have contributed survival; sulfur-containing amino acids, cysteine or cystine, are
usually required for the cultivation of F. tularensis. Sodium chloride also enhances F.
tularensis culture growth. By forming a biofilm  in natural ecosystems,  Francisella might be able
to survive the environmental conditions of mud and waterways, and forming a biofilm could be
a mechanism for persistence within the ticks.

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.
No information  is available on its persistence in soil.

Unidentified constituents in the air (possibly olefins from oil refineries and dense car
populations) may reduce F. tularensis viability. No information in the influence of solar
radiation (UV light) on F. tularensis persistence.

Viral Encephalitis and Hemorrhagic Fever Agents

Encephalitis viruses (category B) and hemorrhagic fever viruses (category A) are viral agents.
Viruses for which persistence was studied include viral hemorrhagic fever filoviruses (e.g.,
Ebola and Marburg) and arenaviruses (e.g., Lassa and Machupo), flaviviruses (Japanese
encephalitis virus, St. Louis encephalitis virus, and yellow fever virus), bunyaviruses
(hantavirus and Crimean-Congo virus), and alphaviruses (Venezuelan equine encephalitis

Japanese encephalitis virus and VEE virus appeared to have inverse relationships with
relative  humidity (RH) in aerosols. Persistence data were only identified for viral hemorrhagic
fever agents in  aerosols and on fomites.

Persistence data on viral encephalitis and hemorrhagic fever agents is sparse. No data were
identified on the persistence of viral encephalitis or hemorrhagic fever agents in soil.
Persistence studies on viral encephalitis  and hemorrhagic fever agents in aerosols or on
fomites  were not continued until attempts to culture produced no viable organism. So, the
longest  duration that these agents could  survive as aerosols or on surfaces  remains unknown.
Studies in water were limited to VEE virus. None of the studies identified for viral encephalitis
and hemorrhagic fever agents assessed  the impact of sunlight on persistence.

 Yersinia pestis

 Y. pestis is a non-spore-forming non-motile bacterial category-A agent, which causes plague.
 Y. pestis can enter a state where they are metabolically active, yet not culturable, with
 unknown viability in water. Y. pestis is also possibly survives inside amoeba. Most persistence
 data on Y. pestis were associated with survivability in water. Y. pestis persistence in water
 ranged from days to years with no apparent relationships between environmental conditions
 and persistence. 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.

 Few studies were found focusing on the impact of various environmental parameters (e.g.,
 humidity, sunlight, and temperature) on persistence. Little data were identified on the
 persistence of Y. pestis in aerosols  and fomites.

 The range of persistence durations  are summarized by agent and medium in Table 1. The
 table also includes the environmental condition associated with each value and identifies
 agent/medium combinations where  persistence data are lacking. Persistence was found to be
 highly variable between species and subspecies.  Persistence was also found to be affected
 by various environmental media and factors (e.g.,  temperature, relative humidity (RH), and
 sunlight), preparation and application methods, and nutrient conditions. Persistence can
 increase in the presence of organisms that serve as hosts or symbionts (e.g., amoeba,
 earthworms); persistence can decrease in the presence of competing and/or predatory
 organisms. Differing analytical methods also affect comparison or interpretation of persistence
 results across studies (e.g., different culture media, incubation temperatures and times).
 Similarly, the estimates of persistence may differ depending on the technique used (molecular,
 culturing, counting, etc.).

Table 1. Agent Persistence in the Environment
Bacillus anthracis*
Brucella species
Shortest Duration Reported
Persistence Associated Environmental
Duration Condition
25C, 80% RH on stainless steel
6 hours coated with silver and zinc zeolite
n_ . . . Topsoil RH 46%, UV and no . .
96 hours(veg) K . ... U.
3 days Distilled water
Galeano et al.
S. EPA, 2014
Sinclair et al.
. , 22C, 45% RH on painted joint
4 nours tape
<4 days Dried soil
<1 day 37C
Ryan (2010)
Environmental survival (specific
3 weeks fomites not identified) in wet,
humid, or dark conditions
. , Dechlorinated municipal water,
1 aay 5C and 25C
Dvorak and

Gilbert and
Rose (2012)
Longest Duration Reported
Associated Environmental
7 , . 37C on polystyrene and glass as
aaysj a biofi|m in BH| broth
40 years (spore)
120 hours (veg)
100 hours(veg)
6 days
Lee et al.
Manchee et al.
Topsoil, RH 46%,no UV U
Topsoil, RH 60%
(approx.) simulated
S. EPA, 201 4c
S. EPA, 201 4c
Sinclair et al.
22C, 40% RH on aluminum and
56 daysf glass; and 5C, 30% RH on
aluminum, glass, and wood
43 days
77 days
Bison partition sites in Greater
Yellowstone, identified in April
Room temperature
Calfee and
Aune et al.
3 months
On stable bedding, troughs,
and harness equipment
Malik et al.
28 days
Tap water, room temperature
Miller et al.

^ Soil
Coxiella burnetii
tularensis Fomite
6 hours
<10 days
60 minutes
Applied in Butterfield buffer to
glass, paper, polyethylene, and
stainless steel; and applied in BHI
broth to stainless steel
Soil inoculated with antagonistic
bacteria (e.g., B. multivorans)
Water exposed to sunlight
Shams et al.
Linetal. (2011)
Sagripanti et al.
20 daysf
20C, 4C, and -20C
Evstigneeva et
al. (2007)
29 minutes
(Tgg value) 
20 minutes
50% RH, wet dissemination
Desiccated on filter paper
Sinclair et al.
(2008); Cox
(1971); Cox and
Goldberg (1972)
Faith et al.
<1 day
Dechlorinated municipal water,
5C and 25C
Gilbert and Rose
,. , Applied in BHI broth to paper,
a^s polyethylene, and stainless steel
Soil stored in plastic bags at
30 months ambient temperature (1 3C to
16 yearsf Distilled water, 25C
Shams et al.
Thomas and
Faulkner (1981)
Pumpuang et
al. (2011)
20 daysf 20C, 4C, and -20C
Evstigneeva et
al. (2007)
5 days gQ% R|_^ wgt dissernination
16 daysf 37C, 0% RH on stainless steel
Sinclair et al.
(2008); Cox
(1971); Cox
and Goldberg
34 daysf Brackish-water, 21 C
Berrada and

Agent Medium
encephalitis and Fomite
hemorrhagic ,., ..
fovo|- agents
Yersinia pestis Fomite
Shortest Duration Reported
1 hourf
5 minutes
22C, 50% RH
Desiccated, 37C
Smither et al.
Fogarty et al.

<60 minutes
34 minutes
(Tgg value)
7 minutes
(Tgg value)
24 daysf
1 day
Tap water (1 mg L1 free
available chlorine or
2 mg L1 total bromine), 21 C
26C, 87% RH
30C, 52% RH on metal
(stainless steel)
Arizona soil during late October
in an area with limited exposure
to UV light
Dechlorinated municipal water,
Wade et al.
Sinclair et al.
Sinclair et al.
Eisen et al.
Gilbert and
Rose (2012)
Longest Duration Reported
120 days
(Tgg value)
50 daysf
Associated Environmental
21 C, 23% RH
4C, 55% RH in tissue culture
medium on glass
Sinclair et
al. (2008)
Piercy et al.
69 days
(Tgg value)
57 minutes
(Tgg value)
7 daysf
10 monthsf
3 yearsf
26C, 50% RH
20C, 50% RH on aluminum
and painted joint tape
Autoclaved river water
Sinclair et al.
Sinclair et al.
Ryan (2010)
Sinclair et al.
Pawlowski et
al. (2011)
BHI, brain-heart infusion; RH, relative humidity; uv, ultraviolet; veg, vegetative state.
- 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).
 The time required for the microbial count to decrease by 99%


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