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EPA/600/R-14/216
September 2014
Literature Review on Mechanisms that Affect Persistence of Bacillus anthracis
in Soils
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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Disclaimer
The U.S. Environmental Protection Agency (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
reviewed by the Agency but does not necessarily reflect the Agency's views. Reference herein
to any specific commercial product, process, or service by trade name, trademark, manufacturer,
or otherwise does not 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; therefore no assurance
can be made that the data extracted from these publications meet EPA's stringent quality
assurance requirements.
Questions concerning this document or its application should be addressed to:
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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Executive Summary
Risk associated with the presence of B. anthracis in the environment in the United States,
depends on a variety of bioclimatic factors such as soil type, environmental conditions, and
ecology, as well as bacterial lifecycle, persistence, and potential to lose (or gain) virulence.
Persistence is the ability of an organism to remain viable (e.g., to remain alive) over time under a
given environmental condition and medium. The purpose of this study was to determine the
current state of scientific knowledge regarding the persistence of B. anthracis spores and
vegetative cells in soil matrices. Information about B. anthracis for this literature review was
considered from reports, peer-reviewed journal articles, books, and government publications
focusing on publications in the last ten years, but including relevant information dating back to
the 1940s from open literature. The agent name and "persistence" or "survival" were used as the
primary search terms, but the terms "viability" and "recovery" were also included. This report
describes a multifaceted B. anthracis lifecycle in combination with genetic, ecologic, and
meteorologic factors that have enabled B. anthracis spores to persist in the environment.
The vegetative form of B. anthracis does not compete well with other soil microorganisms and is
often considered an obligate pathogen, unable to reproduce outside of an infected host in nature.
Establishment of a persistent cycle from a host, to soil, to a host is the basis for recurrent
outbreaks. However, recent work has noted that vegetative cells may persist up to 120 hours in
topsoil and an inoculated mixture of spores and vegetative cells on topsoil persisted up to 56
daysl. The spore possesses numerous protective properties. The outer exosporium layer of the
spore tends to be more hydrophobic, which aids the spore in adhering to the surrounding
environment.
In addition to the stability of vegetative cells, B. anthracis has more recently been found to
survive and grow in non-host microenvironments, which contradicts with the obligate
mammalian pathogen theory. For example:
• B. anthracis was found to propagate in the rhizosphere of a common pasture grass and
evidence of plasmid transfer between two strains of B. anthracis in the model rhizosphere
system was observed2. This finding is significant as it provides evidence of metabolically
active B. anthracis cells in the plant-soil environment. It has been hypothesized that
growth of B. anthracis within rhizome nodules might be the missing environmental
reservoir of B. anthracis between natural anthrax outbreaks3. However, other studies
have found that the presence of the grass does not necessarily increase B. anthracis
1 U.S. EPA, 2014. Environmental persistence of vegetative Bacillus anthracis and Yersinia pestis. EPA/600/R-
14/150.
2 Saile andKoehler, 2006. ApplEnviron Microbiol 12 (5): 3168-3174.
3 Kiel et al. 2009. In Proceedings ofSPIE eds. Fountain, A.W., III and Gardner, PJ. Bellingham, WA
in
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survival or multiplication in the soil, and theorized that the presence of B. anthracis in
soil may promote establishment of grass at carcass sites, thereby potentially increasing
the transmission of B. anthracis to grazing hosts by attracting more grazing animals to
the site4.
• B. anthracis spores have been found to germinate within common soil amoeba and
replicate to the point of lysing the amoeba host. Upon lysis, the vegetative cells will
sporulate in a simulated stagnant water/moist soil environment. Research has also found a
pXOl plasmid dependence for spore germination within the amoeba, which might explain
why a second plasmid associated with infection, pX02, is lacking in multiple natural B.
anthracis strains - it is not required for proliferation5.
• Similarly, an interaction between the intestinal tract of earthworms and B. anthracis has
been shown to be dependent upon the presence of various bacteriophages (viruses that
infect bacteria). Bacteriophage infection of B. anthracis could create lysogens (the
integration of phage nucleic acid into the bacterial chromosome) that restore functional
gene activity necessary to survive and replicate in earthworms, rhizosphere, biofilms, and
soil6.
Soil has been hypothesized to help aid dispersion and protection of B. anthracis spores in the
environment. Spores have been found to move between solid surfaces and water and tend to be
in water7. Spore transport might also occur during flooding, during natural drainage, and due to
reaerosolization of the spores from soil caused by wind.
In an effort to better characterize anthrax distribution throughout the world, researchers have also
been working to develop better methods to characterize occurrence. For example, genomic
analysis techniques have been used more recently to track B. anthracis genetic sublineage in
order to understand genetic-ecological associations and to construct ecological niche models to
predict natural outbreaks and to rapidly identify intentional anthrax events8. However,
identifying B. anthracis spores in an environmental sample continues to be a difficult task due to
the inhibiting compounds within a soil matrix, low concentrations of the spores themselves, and
low processing efficiencies. The lack of efficient methods to isolate B. anthracis spores from soil
has affected the ability of scientists to accurately characterize the persistence of the spores in
4 Ganz et al. 2014. PLOSNeglected Tropical Diseases 8(6): e2903.
5 Dey et al., 2012. Appl Environ Microbiol 78 (22): 8075-8081.
6 Schuch and Fischetti, 2009. PLoS One 4 (8): e6532.
7 Chen et al. 2010. Colloids SurfB Biointerfaces 76 (2): 512-518.
8 Mullins et al. 2011. BMC ecology 11 32; Kracalik et al. 2013, PLoSNegl Trap Dis 7 (9): e2388; Mullins et al.
20l3,PLoS One 8 (8): e72451.
IV
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nature. Work on improving the recovery of B. anthracis from soil and other matrices is
continuing.
Several knowledge gaps, as identified by this review, need to be addressed in order to understand
the human risk associated with B. anthracis residual contamination and persistence of spores in
the environment. The gaps include, but are not limited to, the correlation of non-host
microenvironments on persistence, factors affecting sporulation, location of spores during
dormancy periods, natural attenuation of B. anthracis, and how readily virulence plasmids are
lost and regained in nature.
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Table of Contents
1. Introduction 1
2. Purpose 2
3. Methods 3
4. Results 4
5. Discussion 14
6. References 17
List of Figures
Figure 1. B. anthracis natural lifecycle.
VI
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List of Acronyms and Abbreviations
CPU Colony forming units
DNA Deoxyribonucleic acid
EPA U.S. Environmental Protection Agency
GABRI Ground anthrax Bacillus refined identification
ML Milliliter
MLVA Multiple locus variable tandem repeat analysis
SASP Small acid-soluble proteins
UV Ultraviolet
Vll
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Acknowledgements
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
Worth Calfee
Erin Silvestri
Joseph Wood
Battelle, Contractor for the U.S. Environmental Protection Agency
The following individuals are acknowledged for their review of this report:
U.S. Environmental Protection Agency
Ann Grimm
Marissa Mullins
Eugene Rice
Vlll
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1. Introduction
Anthrax outbreaks have occurred for thousands of years, and have been hypothesized to have
caused the fifth and sixth plagues as described in the Bible, in Exodus, Chapters 7-9 (Hugh-Jones
and de Vos 2002). Robert Koch first described the etiology of anthrax in 1876, and significant
work has been devoted to the organism in the years since (Fennelly et al. 2004; Hudson et al.
2008). Risk associated with the presence of B. anthracis, the causative agent for anthrax, in the
environment in the United States, depends on a variety of bioclimatic factors such as soil type,
environmental conditions, and ecology, as well as bacterial lifecycle, persistence, and potential to
lose (or gain) virulence.
A key consideration in understanding the environmental impact of Bacillus anthracis cells is the
stability of the organism in a variety of matrices. Persistence is the ability of an organism to
remain viable (e.g., to remain alive) over time under a given environmental condition and
medium. While the vegetative form of B. anthracis is not expected to persist in soil as it does not
compete well with other microorganisms in nature (Titball et al. 1991; Hugh-Jones and
Blackburn 2009), when they are exposed to certain conditions, such as contact with air, they
rapidly sporulate to form very persistent spores (Hudson et al. 2008). Once formed, B. anthracis
spores can resist prolonged periods of desiccation, extremes in temperature, pressure, pH,
ionizing radiation, and ultraviolet (UV) radiation, allowing spores to potentially survive for
hundreds of years (Dragon and Rennie 1995).
B. anthracis persistence also depends on chemical and environmental characteristics as well as
surviving the competition from other organisms (Minett and Dhanda 1941; Van Ness 1971). The
There have been numerous examples of extended B. anthracis stability in the environment. For
example, in pond water, B. anthracis spores have been reported to survive 18.5 years (Minett and
Dhanda 1941); in moist or dry soil for years (3 months to 33 months) (Manchee et al. 1981;
Sinclair et al. 2008; Hugh-Jones and Blackburn 2009); in soil or gravel at anthrax carcass sites
for several years to decades (Manchee et al. 1981; Turnbull et al. 1998; Sinclair et al. 2008). B.
anthracis has also been found to survive for 10 to 22.5 years on canvas held at room
temperature, with low humidity and diffuse sunlight and was found to be viable up to 40 years
within dried blood on gauze (Army Biological Defense Research Center 1953). B. anthracis
spores persist best in dry conditions with soils that are relatively alkaline (above pH of 6), high in
calcium, and high in organic matter (Dragon and Rennie 1995; Hugh-Jones and Blackburn
2009).
Anthrax is endemic for livestock living in warmer climates, including the United States (Van
Ness and Stein 1956; Dragon and Rennie 1995). As such, livestock are seen as a primary source
of B. anthracis in the environment. Globally, human outbreaks of anthrax due to environmental
exposure are widespread (Kracalik et al. 2013). Humans are susceptible to anthrax infection from
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three routes of exposure: cutaneous (by introduction of spores into a lesion or by an insect bite
transmitting spores from an infected animal); gastrointestinal (by ingesting contaminated meat or
water); and pulmonary (by inhaling spores) (WHO 2008). Johnson (2007) has developed a list of
criteria to differentiate between natural and non-natural epizootics of anthrax in livestock
populations after an event (an outbreak or an attack) has occurred. Blackburn et al. (2007) has
developed an ecological niche modeling tool to predict the geographical distribution of B.
anthracis across the continental United States prior to observed outbreaks by using historic
wildlife and livestock outbreak data along with several environmental variables. Their study
depicts a significant corridor of increased B. anthracis presence running north to south from
Canada to Mexico. Griffin et al. (2009) was able to confirm the existence of B. anthracis isolates
within a similar transect of North American soils. The identified areas follow historical cattle
trails (Blackburn et al. 2007). In many instances, recent anthrax cases are associated with old
graves of anthrax stricken animals and suitable soil conditions (Pepper and Gentry 2002; Griffin
et al. 2009; Hugh-Jones and Blackburn 2009; Kracalik et al. 2013).
Assessing risk associated with the presence of B. anthracis in the environment in the United
States, whether as background levels, during an outbreak, or as residual levels after an act of
bioterrorism, might depend on a variety of factors such as soil type, environmental conditions,
lifecycle, persistence, ecology, and potential to lose (or gain) virulence. The present study
provides a literature review of the state of knowledge with respect to the persistence of B.
anthracis vegetative cells and spores in environmental soils. Knowledge gaps are also identified
in this review.
2. Purpose
The purpose of this study was to determine the current state of scientific knowledge regarding
the persistence of naturally occurring B. anthracis spores in soil matrices. A discussion of
persistence of B. anthracis vegetative cells has also been included for completeness. Within this
document, persistence refers to spores or vegetative cells that are viable and culturable. The
ability of these cells to subsequently cause infection or induce human disease following release
into the environment is beyond the scope of this review.
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3. Methods
Information about B. anthracis for this literature review was considered from reports, peer-
reviewed journal articles, books, and government publications focusing on publications in the
last ten years, but including relevant information dating back to the 1940s from open literature.
Books were limited to those published or revised in the last 10 years. The primary search engine
used was Google Scholar™ ; Pubmed® and Chemical, Biological, Radiological, and Nuclear
Defense Information Analysis Center were used secondarily. Search terms included the agent
name plus "persistence," "survival," "viability," and "recovery." The review focused on the
environmental persistence associated with soil. The search was limited to articles published in
the English language, but there was no restriction on geographic location. Additional search
terms were used in conjunction with the agent name and "persistence," "survival," "viability,"
and "recovery" as a check to ensure important variables affecting the agent's persistence were
captured including: soil type, geochemical properties of the matrix, rhizosphere, native
organisms, soil pH, weather patterns, moisture, temperature, relative humidity, time, UV light
and solar radiation.
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 U.S.
Environmental Protection Agency (EPA) quality assurance requirements. However, the sources
of secondary data were limited to peer-reviewed documents.
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4. Results
Persistence, sporulation of B. anthracis vegetative cells, and spore structure. B. anthracis
vegetative cell persistence depends on chemical and environmental characteristics and on
surviving competition from other organisms (Van Ness 1971). The vegetative form of B.
anthracis does not compete well with other soil microorganisms in nature (Titball et al. 1991;
Hugh-Jones and Blackburn 2009). B. anthracis forms metabolically dormant and extremely
resistant spores as a survival mechanism when vegetative cells experience nutrient-limiting
conditions (Ghosh and Setlow 2009; 2010). These environmentally resistant spores are
composed of a series of concentric layers that each has a role in extending the persistence of the
organism (Koehler 2009). At the center is the core, where the chromosome along with tightly
bound small acid-soluble proteins (SASPs) are found. High levels of calcium dipicolinic acid
along with the SASPs protect the core deoxyribonucleic acid (DNA) from UV degradation
(Driks 2009). A salt lattice structure formed between the calcium and the dipicolinic acid
stabilize the DNA and enzymes within the core increasing the thermoresistance properties of the
spore (Himsworth 2008). The membrane and peptidoglycan cortex layers surround the core, and
work together to keep the core dry (Driks 2009). Finally, two protein layers called the spore coat
and exosporium surround the cortex of B. anthracis spores (Driks 2009). The coat prevents
foreign materials from entering the core (Driks, 2009). The exosporium is present in several
Bacillus species, including members of the B. cereus group. It is composed of a basal layer and
a hair-like nap layer made up of glycoproteins which interact with the environment to signal
when conditions are optimum for germination (Driks 2009; Kailas et al. 2011; Thompson et al.
2011).
The structure of the exosporium and its nap coat are thought to aid in binding spores to charged
particles in soil, and thereby influence the dispersion of Bacillus spp. spores (Hugh-Jones and
Blackburn 2009). Multiple works show that both soil characteristics and spore exosporium
structures play a role in how well a spore will adhere to its surroundings. Williams et al. (2013)
found that while the presence of the exosporium impacts the adherence to surfaces, the impact is
less noticeable on steel than in soil. Taylor-McCabe et al. (2012) correlated the recovery
efficiency of B. anthracis spores from porous (sand) and non-porous (clay) soils to DNA
signature recovery. They found that, in general, the more hydrophobic the exterior of a spore
strain the better adhesion the spore will have to its surrounding environment. B. anthracis spores
have been found to have higher cell surface hydrophobicity and the least negative electrophoretic
mobility compared to other commonly used surrogates (White, et al. 2012; White et al. 2014). It
has been hypothesized that the high hydrophobicity and more negative charge of the spores in
higher pH ranges (such as alkaline soil) prevents loss of calcium from the spore core and thus
maintain germination capability and viability (White et al. 2012; White et al. 2014). Work
looking at leachate from simulated landfills confirms that the hydrophobic nature of the B.
atrophaeus spores used in the study retarded their transport through the simulated landfills
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(Saikaly et al. 2010). Furthermore, Chen et al. (2010) determined that functional groups on the
spore surface significantly influence transport properties, and thereby also influence the
persistence of B. anthracis spores in nature.
Spore physiology influences the resistance and dispersion of B. anthracis spores. In particular,
the metal ions available during sporulation have been found to influence spore characteristics.
Bacillus spp. spores enriched with calcium germinated more quickly than spores enriched with
strontium or barium (Himsworth 2008).
Initiation and speed of B. anthracis sporulation is also dependent on temperature and relative
humidity (Minett 1950; Hugh-Jones and Blackburn 2009). Minett (1950) reported that
sporulation associated with open carcasses is dependent upon temperature, with sporulation rates
increasing as temperatures increase; at 36°C sporulation occurs in 8 hours, at 32°C by 10 hours,
at 26°C by 18 hours, and at 21°C by 24 hours. The EPA (2014) found that the vegetative cell
population grew by a factor of 10 and had extensive sporulation on wet topsoil within 48 hours
when held at room temperature and high relative humidity. When an inoculum contained B.
anthracis in both vegetative cell and spore form, both cells and spores were recovered from the
test materials 56 days after inoculation. While a temperature of 39°C and high relative humidity
(100%) has been cited as being ideal for sporulation to occur (Hugh-Jones and Blackburn 2009);
sporulation in soil is reported to only occur at temperatures above 25°C (Lindeque and Turnbull
1994). At a temperature of about 20°C, sporulation is inhibited and the vegetative form of B.
anthracis dies (Hugh-Jones and Blackburn 2009).
While the spore possesses numerous protective properties, some studies show that it is still
susceptible to environmental degradation. Laboratory studies conducted by Minett (1941) have
r\
shown that exposure to sunlight can kill a spore within 84 hours. UV radiation at 452 erg/m
(45.2mJ/m2) and 253?A (253.7 nm) destroys 90% of cells (Army Biological Defense Research
Center 1953). However, environmental soils might have a UV A/B protective effect for B.
anthracis cells. EPA (2010) compared the persistence of B. anthracis spores spiked onto topsoil,
glass, bare pine wood, and unpainted concrete that had been exposed to simulated sunlight (UV-
A/B). Results determined that when exposed to simulated sunlight (UV-A/B) there was
essentially no loss in recovery of B. anthracis spores on topsoil after 28 days, while there was
over a 5 log reduction of spores on glass for the same time period (EPA 2010).
In a more recent study, work with topsoil determined that B. anthracis cells did not sporulate and
were culturable for up to 96-120 hours at normal laboratory temperatures and a relative humidity
of 46% regardless of UV-A/B (simulated sunlight) exposure (EPA 2014).
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B. anthracis genetics and persistence. B. anthracis is thought to have been derived from the B.
cereus and B. thuringiensis clade (Okinaka 2006) and is considered to be closely related to
members of the B. cereus group (Klee et al. 2010; Greenberg et al. 2010). B. cereus is a
saprotrophic, opportunistic pathogen commonly found in the rhizosphere and as a gut
commensal (Schuch and Fischetti 2009), while B. thuringiensis is a common insect pathogen that
has been utilized for decades as a natural pesticide (Tilquin et al. 2008; Van Cuyk 2011). Each of
these organisms share a core set of genes that support growth within a diverse range of
environments. The transcriptional regulator/>/£/? regulates over 100 loci, which allows
organisms such as B. cereus and B. thuringiensis to respond to the environment (Schuch and
Fischetti 2009). However, although the loci are encoded for in B. anthracis, a single nonsense
mutation has caused an inactivation ofplcR and transcriptionally silenced these loci in B.
anthracis, thus allowing the capacity for survival in the environment (Schuch and Fischetti
2009). It is thought that all members of the B. cereus group share a pan genome within which
select portions, specifically prophages, are exchanged through horizontal gene transfer (Klee et
al. 2010). Thus, B. anthracis carries the genetic capacity for environmental survival and
transferred prophages could modify the expressed phenotype to promote environmental
persistence (Schuch and Fischetti 2009).
In an effort to stay at the forefront of anthrax distribution throughout the world, researchers have
begun using genomic analysis techniques to track B. anthracis genetic sublineage (Aikembayev
et al 2010; Kenefic et al. 2008; Mullins et al. 2013). The hope is that by understanding genetic-
ecological associations, adequate ecological niche models can be constructed to predict natural
outbreaks and rapidly identify anthrax events begun from illicit mechanisms (Mullins et al. 2011;
Kracalik et al. 2013; Mullins et al. 2013). While B. anthracis has a limited global diversity,
multiple locus variable number tandem repeat analysis (MLVA) systems can differentiate B.
anthracis into lineages and sublineages (Mullins et al. 2011) and single nucleotide repeat
markers can be used to give a detailed analysis in an outbreak using a fine scale resolution
(Kenefic et al. 2008). Global studies of B. anthracis have shown that the A lineage is globally
distributed, while the B and C lineages are more limited in geographic distribution (Van Ert et al.
2007). The adaptive differences between the three lineages might have aided in the dispersion
and persistence of B. anthracis worldwide.
A comparison of A and B lineage isolates collected from Kruger National Park, South Africa
found that the B strains were only found in soils with significantly higher calcium concentrations
and soil pH measurements when compared to the A strains (Smith et al. 2000; Hugh-Jones and
de Vos 2002). In fact, the large genetic differences found among samples collected from Kruger
National Park have led some to hypothesize that southern Africa might be the geographic origin
of B. anthracis (Smith et al. 2000). Genotyping work in the Central Asian nation of Kazakhstan
shows that B. anthracis Al.a sublineage was associated with a more diverse ecologic distribution
than the other A lineage isolates studied (Mullins et al. 2011). Furthermore, the Al.a sublineage
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is the prevalent B. anthracis lineage across the United States, Italy, and Kazakhstan (Van Ert et
al. 2007; Aikembayev et al. 2010; Fasanella et al. 2010; Mullins et al. 2011). Results of
ecological niche modeling studies characterizing the Al.a sublineage across theses three
countries suggest that specialization within the Al.a sublineage might have occurred over time
(Mullins et al. 2013). This genetic specialization for various geographic locations has pointed to
the need for regional ecological niche models that account for genetic diversity of the B.
anthracis lineage(s) present in conjunction with soil and climate characteristics (Mullins et al.
2013). Such knowledge has recently led researchers to determine that an anthrax outbreak in
Bangladesh was most probably due to the domestic recycling of bone as bonemeal for livestock
feed rather than contaminated soils. Fasanella et al. (2013) recovered three distinct MLVA
genotypes of the sublineage A.Br.001/002 from six geographically close herds that primarily stay
on clay soils. Work by Ahsan et al. (2013) furthered the findings by Fasanella et al. (2013) by
collecting additional soil samples from low lying areas, livestock pastures, and burial sites near
the original sampling sites. Fourteen of the 48 samples collected by Ahsan et al. (2013) were
positive for B. anthracis spores. None of the clay samples were positive for B. anthracis, but
rather all of the spore positive samples were from loamy soils with an elevated moisture content
(16.69±2.06%) and slightly acidic pH (6.38±0.15) (Ahsan et al. 2013). Kenefic et al (2008)
found multiple SNR genotypes and a random spatial and temporal pattern of isolates of B.
anthracis after taking blood and tissue samples from 47 cattle that were associated with an
outbreak in South Dakota in 2005. His results suggested that the area was associated with
multiple past outbreaks and conditions that favored spore survival. Together these reports
indicate that while anthrax could be occurring at a higher rate due to contaminated feedstock, the
area also has a natural propensity to harbor B. anthracis spores in select areas.
B. anthracis lifecycle and environmental persistence. Members of the Bacillus genus are
primarily saprophytes (organisms that obtain nutrients from dead matter) living in soil (Saile and
Koehler 2006). In contrast, B. anthracis has traditionally been considered an obligate pathogen,
unable to reproduce outside of an infected host in nature due to a single nonsense mutation that
inactivated a transcription regulator gene attributed to saprophyte capabilities (WHO 2008;
Schuch and Fischetti 2009). Establishment of a persistent cycle from a host, to soil, to a host has
been seen as the basis for recurrent outbreaks (Van Ness 1971). The classic B. anthracis lifecycle
(Figure 1) involves:
• a period of prolific growth (on the order of multiple millions of vegetative organisms per
milliliter of blood within an infected individual, producing toxins that kill the host)
• rapid (condition dependent) sporulation at the carcass site initiated when predation (or
other events) open a carcass, allowing the bodily fluids to drain from the infected carcass
• dispersion of vegetative cells into the environment (air, soil, or water)
• spore acquisition by a new host
• spore germination in a new host (Lindeque and Turnbull 1994; Schuch and Fischetti
2009)
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B. anthracis is present at high levels, a million to a billion CFU/mL in the blood of animals when
they die of anthrax (Lindeque and Turnbull 1994). High carbon dioxide concentrations that are
found in decomposing carcasses reduce sporulation. B. anthracis in carcasses die in four days or
less under conditions supporting anaerobic digestion (Minett 1950; Hugh-Jones and Blackburn
2009) and under conditions where they are in competition with other microorganisms (Dragon
and Rennie 1995). For sporulation to occur, carcasses must be opened, or bodily fluids drained
from the carcass (Minett 1950; Johnson 2007; Hugh-Jones and Blackburn 2009). Predators are
responsible for opening carcasses, and thereby facilitating sporulation (Dragon et al. 2005). In
locations and/or conditions not conducive to sporulation, spilled blood of infected animals with
high B. anthracis colony forming units (CPU) per milliliter (mL) could result in few or no spores
in soil as vegetative B. anthracis survival outside of a host is poor (<24 h) (Lindeque and
Turnbull 1994; Atlas 2002).
Anthrax outbreaks have been known to stop occurring for decades at a site before an herbivorous
(typically) host again becomes infected and the cycle is repeated (Hugh-Jones and Blackburn
2009). Details of natural B. anthracis cycles during the long dormancy periods and explanations
for the initiation of outbreaks after dormant periods are incomplete and controversial. For an area
to provide a risk of anthrax, virulent B. anthracis must be present. B. anthracis is most often
found in dry conditions with soils that are high in organics and calcium, and are relatively
alkaline (above pH 6) (Van Ness 1971; Hugh-Jones and Blackburn 2009; Aikembayev et al.
2010). In a study that aimed to map the spatial and temporal distribution of anthrax outbreaks in
Kazakhstan, foci for anthrax were concentrated more in regions with alkaline soil and higher
organic matter (southern and areas in northern regions) compared to areas that consisted of desert
and poorer soil conditions (central Kazakhstan) (Aikembayev et al. 2010). In a review by
Sinclair et al. (2008) it was noted that the lifecycle of B. anthracis includes a soil dwelling stage,
and that viable spores can be recovered after significant periods (40, 60, and potentially 1,000
years). However, while the potential for germination and multiplication of B. anthracis in the
soil dwelling stage were mentioned, the mechanism and conditions favoring this germination
were not discussed in the Sinclair et al. (2008) review.
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Vegetative Form
Spore form
INFECTED ANIMAL HOST
host disease
and death
V
CONTAMINATED
CARCASS
release of
anthrax with
fluids from
carcass
v
SOIL
germination
sporulation
ANIMAL HOST
exposure
of animal
host to
spores
SOIL OR NATURAL
ENVIRONMENTAL
RESERVOIR(S)
Figure 1. B. anthmcis natural lifecycle; modified from Schuch and Fischetti (2009).
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The concept that spores germinate and propagate in soil is not new. West and Burges (1985)
spiked spores of B. thuringiensis and B. cereus into sandy silt loam soils supplemented with
either grass clippings or chicken manure. Their results showed that B. thuringiensis spores
germinated and propagated in 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. Similarly, B. thuringiensis and B. cereus spores germinated in
manure-supplemented soil; however, after the initial burst of activity the viability counts
significantly decreased (West and Burges 1985). These results indicate that though spores might
be activated by an abundance of fresh nutrients in supplemented soils, germinated cells do not
readily persist in all soils (West and Burges 1985). Other works have also suggested that B.
thuringiensis can proliferate in nature under ideal conditions. Tilquin et al. (2008) found that B.
thuringiensis subspecies israelensis spores, originally sprayed in France as a means of mosquito
control, were found in high concentrations in leaf matter (3 x 105 spores g"1). The authors suggest
that the high concentrations of B. thuringiensis found might be because the leaf litter is a specific
microenvironment in which the organism can persist and grow. Specifically, the work
highlighted two key elements to the microenvironment that potentially contributed to the
increase in spore counts- low oxygen levels and low decomposition rates (Tilquin et al. 2008).
While these studies did not utilize B. anthracis, their work is important because both B.
thuringiensis and B. cereus are routinely used as surrogates for B. anthracis.
B. anthracis dispersion and persistence in nature. It has been hypothesized that soil might aid
in both dispersing and protecting B. anthracis spores in nature (Williams et al. 2013). Within this
theory, soil surrounding the spores limits UV A/B radiation exposure (EPA 2010), while spore-
soil binding influences spore dispersion patterns. Work by Chen et al. (2010) determined that B.
anthracis Sterne spores are monopolar and negatively charged. These findings indicate that in
natural environments where spores can move between a solid surface and water (i.e. in soil near
a body of water), spores will tend to be in water much of the time (Chen et al. 2010). This
conclusion corroborates findings by Kim et al. (2009) who determined that an increase in water
flow rate has the greatest effect on spore movement. Not only will spores tend to be in water, but
also as the water velocity increases, hydrodynamic forces acting on the soil surface increase,
which results in the decrease of interparticle bonding forces and increased spore movement.
Thus, periods of local flooding which raises the water table could detach spores from their refuge
and transport them back to the soil surface, contaminating the vegetation grazed by susceptible
herbivores, and thus begin the B. anthracis lifecycle again (Kim et al. 2009; Williams et al.
2013).
Spore transport during local flooding and natural drainage could also account for limited
horizontal dispersion patterns (Manchee et al. 1994; Hendriksen and Hansen 2002; Hugh-Jones
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and Blackburn 2009; Kim et al. 2009; Williams et al. 2013) and areas of higher spore
concentrations in the environment (Van Ness 1971; Dragon et al. 2005; Griffin et al. 2009;
Williams et al. 2013). Van Ness (1971) hypothesized that spores germinate and proliferate
following a flood event then quickly re-sporulate. Dragon and Rennie (1995) called areas of soil
with high spore concentrations "storage areas." In their assessment, rather than reproducing,
hydrophobic spores were transported to low-lying areas where they were concentrated as the soil
dried out and left to interact with plant cuticles, increasing exposure of grazing animals to the
spores (Dragon and Rennie 1995). While a connection between local flooding and anthrax
incidence has been recognized, the exact role in dispersion and persistence of B. anthracis spores
is still uncertain.
The role wind and ground perturbation plays in dispersing B. anthracis spores from soils has
been assessed (Layshock et al. 2012). In 1949, military researchers sprayed grassy fields with
either a dry powder or wet slurry of B. anthracis before collecting air samples while troops
marched through the fields. Their results showed minimal reaerosolization from the troop
activity (Peck 1949). Aerosol samples have also been collected downwind from anthrax carcass
sites (Turnbull et al. 1998). It was found that B. anthracis spores were only found downwind of
the carcass site during periods of manual disturbances and with the highest wind gusts. These
results led Turnbull et al. (1998) to conclude that spores are mostly attached to large soil
particles, and do not remain airborne for very long. Work conducted by Byers et al. (2013) in a
laboratory controlled ambient breeze tunnel determined that the reaerosolization rates of spores
are dependent upon the humidity, temperature, and particle size.
B. anthracis and non-host microenvironment effect on environmental persistence.
Several studies have focused on the ability of B. anthracis to propagate in soil environments. In
one such work, B. anthracis was found to propagate within the rhizosphere of a common pasture
grass (Saile and Koehler 2006). The authors proposed that the plant roots enhanced germination
and proliferation of vegetative cells, as nearly 50% of the inoculated spores germinated in the
presence of plant roots, whereas few to no spores germinated in the absence of the grass (Saile
and Koehler 2006). Work conducted by Kiel et al. (2009) has demonstrated the ability of B.
thuringiensis to grow within rhizome nodules of fescue grass, and a hybridizing relationship
between a barley flavoprotein gene and a nitrate reductase gene of B. anthracis. Studies have
also found evidence of plasmid transfer between two strains of B. anthracis within the model
rhizosphere system (Saile and Koehler 2006). This finding is significant as it provides evidence
of metabolically active B. anthracis cells in the plant-soil environment. Together these findings
have led Kiel et al. (2009) to hypothesize that growth of B. anthracis within rhizome nodules
could be the missing environmental reservoir of B. anthracis between natural anthrax outbreaks.
However, conflicting results were found by Ganz et al. (2014) who aimed to test whether
interaction of B. anthracis, native grass (Enneapogon desvauxii\ and carcass blood increased B.
anthracis survival in soils from Etosha National Park. The results suggested that while B.
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anthracis might increase the transmission of B. anthracis to grazing hosts by promoting
establishment of grass at carcass sites, which attracts the hosts, no evidence was found that the
presence of the grass increased B. anthracis survival or multiplication in the soil (Ganz, 2014).
There have been a number of studies looking at potential commensal or symbiotic relationships
between Bacillus species and soil borne organisms, including amoeba, nematodes, earthworms,
and bacteriophages. Dey et al. (2012) simulated a stagnant water/moist soil environment to
assess the interaction of B. anthracis (Ames and Sterne) with Acanthamoeba castellanii, a
common soil amoeba. The work 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 sporulated in the simulated stagnant water/moist soil environment. Their work
also highlighted the importance of the pXOl plasmid. Virulent B. anthracis contains two
plasmids, pXOl and pX02. Interestingly, a pXOl plasmid was required for spore germination
within the amoeba (Dey et al. 2012). Similar findings were noted in soil samples collected in
New Orleans after Hurricane Katrina. Of the five B. anthracis positive samples collected post-
flood, all had the pXOl plasmid and only one contained both pXOl and pX02 plasmids (Griffin
et al. 2009). Researchers hypothesized that this germination dependence could explain why pX02
is lacking in multiple natural B. anthracis strains - it is not required for proliferation (Dey et al.
2012). There is some evidence that virulence plasmids can be acquired or reacquired to induce
virulence. For instance, B. cereus induced an anthrax-like disease in multiple chimpanzees in the
Ivory Coast. Both pXOl and pX02 virulence plasmids were found in the B. cereus genome in
these animals (Klee et al. 2010). Researchers hypothesized that the virulence plasmids were
obtained from B. anthracis cells in the soil or another animal that was co-infected with B.
anthracis and B. cereus (Klee et al. 2010).
The intestinal tract of worms has been explored as a potential site of B. anthracis spore
germination. Laaberki and Dworkin (2008) fed B. anthracis Sterne spores and vegetative cells to
laboratory-controlled nematodes. They found that the digestive track of the worms killed the
consumed vegetative cells, but spores were able to pass through their system into their feces and
remain viable. This conclusion is supported by work conducted by Hendriksen and Hansen
(2002). However, Hendriksen and Hansen (2002) were able to determine that consumed B.
thuringiensis spores germinated in the gut of the earthworms prior to re-sporulation and
defecation. Furthermore, their study showed that B. thuringiensis was able to germinate in the
hindgut of three of the four tested earthworm species, indicating that this is not a species limited
occurrence, but rather a widely distributed capability (Hendriksen and Hansen 2002).
More recently, the interaction between earthworms and B. anthracis has been shown to be
dependent upon the presence of various bacteriophages (viruses that infect bacteria).
Bacteriophage infection of B. anthracis could create lysogens (the integration of phage nucleic
acid into the bacterial chromosome) that restore the functional gene activity necessary for the
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bacteria to survive and replicate in earthworms, rhizosphere, biofilms, and soil (Schuch and
Fischetti 2009). These lysogens appear to induce phenotypic changes to the B. anthracis
vegetative cells that alter their capacity to sporulate, produce exopolysaccharide for biofilm
production, and survive long-term in soil and in the anoxic earthworm gut (Schuch and Fischetti
2009; Schuch et al. 2010). Research has 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). Work by Schuch et al. (2010) showed
that one bacteriophage, worm intestinal phage 1, was present in high numbers for three years in
Pennsylvania forest soil before the population was replaced by a second bacteriophage, worm
intestinal phage 4, for the next three years of the study. This demonstrates that the bacteriophage
population in environmental B. anthracis-\ike isolates in earthworm intestinal tracts is variable.
Another point of interest is the comparison of the earthworm lifestyle to what is known of the B.
anthracis lifecycle in soils. Both earthworms and B. anthracis prefer alkaline soils with high
calcium concentrations and rich in organics (Hugh-Jones and Blackburn 2009; Schuch et al.
2010). This lifestyle correlation might be an indication as to why significant spore spreading is
not seen in some locations. For instance, after 40 years the spore spread in the acidic soils of
Gruinard Island was limited to the top 6 cm of soil surrounding the original detonation point
(Manchee et al. 1981; Manchee et al. 1994; Sharp and Roberts 2006). Furthermore, even the
limited horizontal distribution seen after 40 years was attributed to wind and/or drainage from
the contaminated areas, not soil borne organisms (Manchee et al. 1994; Hugh-Jones and
Blackburn 2009). Anthrax events also seem to occur more commonly after seasonal flooding
events when earthworms seek the soil surface (Griffin et al. 2009; Schuch et al. 2010). The
combination of lifestyle patterns, hindgut colonization, and anthrax occurrence patterns points to
a significant relationship between anthrax incidence 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.
B. anthracis biofilms and persistence. Under appropriate conditions, B. anthracis readily forms
biofilms (Lee et al. 2007; Auger et al. 2009; Schuch and Fischetti 2009). Biofilms are complex
communities of microbes that produce glycocalyx polysaccharides to protect them from
desiccation, chemical treatment, immunological attack, and antibiotics (Lee et al. 2007; Auger et
al. 2009; Perkins et al. 2010). Biofilms are well known to harbor infectious organisms, lengthen
persistence, and aid in dispersion through sloughing of fringe layers (Perkins et al. 2009). Due to
their structure, biofilm microenvironments have been hypothesized to create a more suitable
environment for vegetative B. anthracis to flourish and engage in genetic transfer in nature (Lee
et al. 2007).
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As mentioned previously, the ability of B. anthracis to form biofilms could have a profound
impact on environmental persistence and proliferation. Biofilm formation can create areas of
nutrient limitation, thereby triggering spore formation for the B. anthracis cells (Lee et al. 2007).
It has also been found that when biofilms are supplemented with carbon dioxide during culture,
the vegetative cell concentration is higher compared to ambient temperature and pressure
conditions (Lee et al. 2007). Studies have shown that B. anthracis strains involved in gut
colonization are adept at forming biofilms (Auger et al. 2009). This finding again points to the
symbiotic relationship between Bacillus species and soil borne organisms.
B. anthracis sampling methods and persistence. Identifying B. anthracis spores in an
environmental sample can be a difficult task due to inhibiting compounds within a soil matrix,
low concentrations of the spores themselves, and low processing efficiencies. Such sampling
difficulties might be one factor for seemingly conflicting results from field sampling efforts.
Multiple detection methods have been developed for clean samples, such as, culture analysis,
biochemical analysis, genetic analysis, and immunologic analysis (Rao et al. 2010; Irenge and
Gala 2012). However, without appropriate sample processing, the most sensitive detection
method will be ineffective. Environmental detection limits for B. anthracis spores in soil have
been estimated to range between 0.1 CFU/g to 3.2xl08 CFU/g soil depending upon the approach
used to evaluate the environmental limit of detection although limited information is available on
which is the best approach (Herzog et al. 2009; Chikerema et al. 2012). As pointed out in a
review by Lim et al. (Lim et al. 2005) there is a need for a universal sample processing method
to separate, concentrate, and purify target agents from any sample type. The lack of efficient
methods to isolate B. anthracis spores from soil has affected the ability of scientists to accurately
characterize the persistence of the spores in nature. A fully developed method with a known
method recovery rate and associated confidence intervals would be useful for determining the
persistence of B. anthracis spores, their viability, and the extent of their presence in the
environment.
5. Discussion
As summarized in this report there are numerous concepts regarding how and where B. anthracis
spores persist in the environment. The evidence included here points to multiple specific
microenvironments that allow B. anthracis with suitable genetic phenotypes to germinate and re-
sporulate in the environment, and thereby both increase in concentration and allow for genetic
transfer (Saile and Koehler 2006; Lee et al. 2007). These microenvironments include amoebae,
biofilms, earthworm intestinal tracts, and grass rhizospheres (Saile and Koehler 2006; Lee et al.
2007; Schuch et al. 2010; Dey et al. 2012). Spores generated through these microenvironments
might be sufficient to carry the species until suitable conditions are met for prolific germination,
such as in nutrient supplemented soils or a mammalian host (West and Burges 1985; Tilquin et
al. 2008; Schuch and Fischetti 2009). Cell concentrations might then quickly dissipate after
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environmental conditions are no longer favorable (West and Burges, 1985). Between anthrax
incidents, spores generated through various microenvironments might be concentrated in the soil
by local flooding events (Kim et al. 2009; Williams et al. 2013). The ability of B. anthracis to
propagate in these microenvironments suggests that it is not an obligate mammalian parasite,
however, more information is needed to make this determination.
While much of the research of these alternative vegetative pathways for B. anthracis has been
conducted in laboratory settings, the fact that earthworms, grasses, and B. anthracis spores all
prefer organic-rich alkaline soils with significant calcium concentrations supports the results of
laboratory findings (Schuch and Fischetti 2009; Joyner et al. 2010). A lack of confirmatory field
sampling might be attributed to inefficient sampling methods for environmental aliquots with
low B. anthracis concentrations and high background organism concentrations (Lim et al. 2005).
More work focusing on environmental soils with its associated invertebrates and rhizosphere are
needed to elucidate the role these identified alternative vegetative pathways have in harboring
and distributing B. anthracis spores between outbreaks.
The studies highlighted herein point to a combination of genetic, ecologic, and meteorologic
factors that allow B. anthracis spores to survive in the environment. In one recent study, Kracalik
et al. (2013) concluded that ecological factors and anthropogenic factors (lifestyles and farming
practices) have a significant role in the persistence of B. anthracis. There has also been
speculation that human efforts to make land more fertile might contribute to anthrax persistence
(Van Ness and Stein 1956). To this end, the Canadian Food Inspection Agency has rescinded its
recommendation of using lime (calcium oxide) as an agricultural anthrax disinfectant as lime
could aid the long-term preservation of B. anthracis spores by providing ample calcium
concentrations (Himsworth 2008).
Knowledge gaps in the available literature make it difficult to extrapolate how and where B.
anthracis persist in environmental soils. These knowledge gaps need to be addressed in order to
understand the human risk associated with B. anthracis residual contamination and the
persistence of B. anthracis spores in the environment. Knowledge gaps identified in this
literature review include:
• Sporulation: What environmental factors are required for sporulation? What is the rate
of sporulation under various bioclimatic conditions?
• Dormancy: Where are B. anthracis spores during anthrax dormancy periods and what
interactions occur to break its dormancy? How does anthrax dormancy affect spore
persistence?
• Exosporium: What role does the spore exosporium structure play in the long-term
persistence of a spore?
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Natural attenuation: What causes or prevents natural attenuation of B. anthracis in the
environment?
Ecology: What is the interaction between bacteriophages, worms, and rhizosphere that
can induce long-term persistence of B. anthracis in soil? How many soil organisms
harbor B. anthracis spores in their gastrointestinal system? What is the distribution of
organisms in various soil types across the United States?
Lysogens: What role do bacteriophage induced lysogens have in the natural B. anthracis
reproductive cycle?
Virulence retention: How readily are virulence plasmids lost and regained in nature?
Biofilms: What role do environmental biofilms play in the long-term persistence of B.
anthracis!
Detection methods: What is the best method for detecting B. anthracis in soil? What is
its limit of detection for multiple soil types?
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