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Burkholderia pseudomallei

Double Paper
Nathan Cain
Micrbiology
Biological Warfare

The use of biologicals as offensive weapons has long been a dirty secret by the nations that used
them. However, the trepidition associated with ‘under-handed’ tactics has seldom prevented people and
states from using organisms both natural and later crafted. “More than two thousand years ago Scythian
archers dipped arrowheads in manure and the flesh of rotting corpses”(9) During the 14th century, “the
Crimean town of Kaffa was captured when the besieging Tatar army catapulted the bodies of plague
victims into the city; the Russians are said to have used similar techniques against the Swedes in the
eighteenth century.”(4) Later, during World War I, German spies were accused of infecting horses and
mules traveling to the front with glanders.(3) However, the true beginning of biological agents as a
method of warfare began with a slight, bespectacled army major, Shiro Ishii. An individual of powerful
drive, Ishii believed the inclusion of biological agents in the Geneva Protocol highlighted the effectiveness
of such agents. Ishii’s beliefs led to the development of the most extensive biological warfare program yet
seen by man. The man himself highlighted the dichotomy of using such weapons. After being captured
by the Americans and asked of the Emperor’s involvement, Ishii is reported as saying,”Biological warfare
is inhumane and advocating such a method of warfare would
defile the virtue and benevolence of the Emperor.”(4) The
Japanese efforts would lead to an oft ignored race during the Cold
War. With advances in technology, and the resources of a
superpower, both the USSR and the USA formed biological
weapons programs in the interest of defense. At Biopreparat, the
goal was to use technology to make stronger, deadlier organisms
that could be used to further Communism. While some of the
stories told by defectors may have been produced by imagination,
several were stated to be theoretically possible such as a
smallpox/Marburg hybrid.(9) However, the threat of a superpower
controlled pandemic has waned for the moment. Richard Nixon
ordered the dismantling of any US offensive capability and Yeltsin
did likewise for the Russian program. The threat now resides in
rogue states and organizations not affiliated with any nations. In
the Dalles, Oregon numerous people were hospitalized with
Salmonella. The year was 1984 and a cult known as Rajneeshee
had released the organism into ten salad bars throughout the
town. More than 750 people fell ill with many being hospitalized
with no casualties. The goal of the group was to influence a local
election and the attack was the first large-scale use of a biological
agent on American territory.(9) In 1995, another cult known as Aum Shinrikyo released sarin gas into the
subways of Japan. They had previously made ten failed attempts to release anthrax. They had
additionally attempted three attacks with the biological toxin botulinum.(9) While these attempts all failed
in the lead up to their use of Sarin, they showed that while not always successful, a dedicated group can
and has been able to acquire the means to use biological weapons. The threat at the time was lessened
due to a lack of knowledge. The same can no longer be said with the presence of rogue nations and the
dispersal of Soviet era scientists. A former member of the IRA once said, “you have to be lucky all the
time, we have to be lucky just once.”(9) The truth of that statement arose in 2001 when an anonymous
letter was sent to legislators and members of
the media. These letters warned that the
perpetrator(s) had anthrax and could not be
stopped. While threats to members of
government and even prominent members of
media could be considered normal, the other
contents of the letter were not. The letters
contained highly purified anthrax spores.
Twenty people contracted the disease with five
of the individuals dying. The death toll would
have been worse had one physician not
recognized the signs and taken appropriate
action to notify the authorities. The anonymity of most biological warfare agents are just one of the many
reasons why an attack using such methods may be so devastating. Steps have been taken since to
insure there are protocols that can prevent similar occurences. Yet as science and knowledge progress,
the task is more and more daunting and new pathogens are emerging every day.

Burkholderia pseudomallei
Burkholderia pseudomallei is a gram negative, facultatively
anaerobic bacillus that causes a condition known as Melioidosis.
The most common morphology grows in a ‘cornflower head’
shaped colony on numerous media.(8) The organism is most
prevalent in Southeast Asia and Northern Australia however the
range of this bacterium has been spreading recently. Cases have
been reported in Africa and the Middle East and in a few areas in
the Western Hemisphere such as “Mexico, El Salvador, Panama,
Ecuador, Peru, Guyana, Puerto Rico, Martinique, Guadeloupe,
and most frequently, Brazil.”(6) B. pseudomallei is in some
regions so prevalent that contamination by the organism in lab
cultures is a common occurrence. The organism is found in soil
and water. The bacillus most often infects equine farm animals
and is the originator of Burkholderia mallei which causes glanders
among humans and animals. While most commonly found in
animals, the condition of Melioidosis is now “considered endemic
in Papua New Guinea, most of the Indian sub-continent, and
southern China, Hong Kong, and Taiwan. This organism
possesses numerous virulence factors that make it extremely
dangerous but also highly unpredictable.

The Great Mimicker


The symptoms of Melioidosis are vastly different among individuals and still do not have a clearly defined
incubation time. In one instance, the incubation period was actually recorded at 62 years.(1) Clinical
presentations are often mistaken for Tuberculosis or varying forms of pneumonia.(6) In perhaps one of
the most worrisome situations, an infection caused by B. pseudomallei was misidentified not just by the
physician but also by an automated identification system.(2) A 35 year old Swiss man was admitted to
the hospital with a cranial abscess as well as defect in the underlying bone. The patient had been to
traveled to southern Asia and could not identify having received an injury to the head. Swelling in the
right parietal area of the head began 17 days after he returned to Switzerland with the bulge showing
constant increase. Puncture showed no aspirate but 7 weeks later the bulge became painful and pus was
present. Tests were performed and samples
grown on sheep’s blood agar. A suspension
was introduced to a Phoenix Automated
Microbiology System which returned an
analysis of B. cepacia with 99% confidence.
The patient was given a preliminary
diagnosis and discharged with a prescription
for Cotrimoxazole. However, additional tests
were run due to a B. cepacia diagnoses
requiring further molecular testing and the
uncommon nature of an abscess being the
site of infection. The patient later returned
for surgery after detailed molecular analysis
disproved the earlier diagnoses. The failure
to properly identify the organism was due to
B. pseudomallei not being present in the
machine’s database. This situation has also
been discovered in the Vitek2 system.(2)
While certainly an unfortunate, and hopefully
soon corrected mistake, the example highlights the difficulties presented by Melioidosis. In the western
hemisphere and especially the developed world, B pseudomallei is an uncommon organism. Additionally,
the variability of symptoms comes into play. Infection can range from acute or chronic localized infection,
to multiple organ septicemia, and often septic shock(1). Ulcers and abscesses may be present as well as
fever and muscle aches. Pulmonary infection can appear to be mild bronchitis or severe pneumonia and
chest X-ray can show cavitary lesions similar to tuberculosis. The condition can produce septicemia
leading to septic shock and/or multiple organ abscess.(6) Antimicrobial therapy is usually a 12-20 week
process and in 10% of patients relapse still occurs.(8)

Virulence

B. pseudomallei is an organism with enormous pathogenic potential. Numerous factors contribute to the
virulence of the organism and in most every area the bacterium locates in the host, there will be a harmful
reaction. The initial danger of B pseudomallei can be found in the extreme diversity within the organism
itself. The organism possesses two distinct chromosomes and roughly 16 genomic islands.(5) In one
study, 3 bacteriophages were isolated from B pseudomallei strains and these strains then produced a
total of 5 distinct phages. These phages were genetically identified as having previously been of the
order Caudovirales and the family Myoviridae (and in 1 case Siphoviridae). More importantly however,
these phages could be closely linked to virulence factors present in other organisms.(7) The
bacteriophages had been subsumed by the bacterium and used to either grant virulence or bolster certain
metabolic characteristics such as toxin-antitoxin modules or iron transport and acquisition.(7) Further, B
pseudomallei cultures occasionally develop colonies of more than one distinct morphology.(8) One study
in particular established the presence of three distinct morphological strains exhibiting an ability to survive
in differing environments. Parental strain I was more resistant to certain chemical methods such as H2O2,
type II formed a smaller rough colony but produced increased amounts of biofilm, and type III was
characterized by increased flagella expression and resistance to antimicrobial peptides. The most
impressive quality of the variable morphologies was the bacterium’s ability to switch between them
dependent on environmental conditions.(8)
With the incredible adaptiveness of B
pseudomallei explored, there are certain
factors that may be considered common to
the species as a whole. The bacillus has
the potential for amazing mobility. As
stated earlier, the organism often presents
with a flagella that allows locomotion both
within a host as well as the environment.
The exterior of the pathogen contains both
capsular polysaccharides as well as
lipopolysaccharides.(5) In many cases
presumably due to the presence of
lipopolysaccharides, an overwhelming
immune response can lead to tissue
damage of the host while the capsule is a
necessary component for survival within the
blood stream.(1) The organism has
multiple natural immunities to antimicrobial drugs due to multidrug efflux pumps that confer resistance to
aminoglycoside and macrolide antibiotics.(5) Once a host is infected B. pseudomallie is capable of
surviving and escaping phagocytosis. This is accomplished by inhibiting NO production from within and by
using T3SS injection into cytosol to lead to cellular structure manipulation.(1) This method may also be
used to gain entrance into nonphagocytic cells.(5) From this point the ability to manipulate actin strands
allows the bacterium to both replicate and move intracellularly. An additional ability exhibited by cells
infected by the bacterium is a process of cell fusion(10). A unique ability of the B pseudomallei, the
organism will induce cells to join into multinucleated giant cells that act as internal reservoirs for the
infection. Finally, the organism has been shown to produce both bio films as well as adhesion proteins
and fimbriae(5).
Infection
In the normal cycle of infection exhibited
experimentally by B pseudomallei, the initial response
to the presence of the bacterium is an overwhelming
inflammatory response. As one study put the effect,
tissue damage was a result of an inflammatory
cascade that was in fact induced and encouraged by
the organism.(1) In mice, there was a release of
cytokines responsible for pro-inflammatory effects.
This was the case up to 24 hour post infection where
the increased inflammatory response led to
macrophage destruction. This effect is shared by
several Gram-negative bacteria such as
Pseudomonas aeruginosa and Legionella
pneumophila. At roughly 16 hours post infection,
ubiquitin D genes would be significantly induced as
well leading to further cell death. Following the initial
24hr period, there would be a shift to the suppression
of various metabolic pathways. Of particular
significance would be the down-regulation of specific
enzymes that lead to the conversion of pyruvate into
acetyl-CoA. Pyruvate dehydrogenase beta production would be inhibited but also alternative pathways
such as fatty acid metabolism, tyrosine metabolism, and various degradation pathways.(1) While the
specific reactions that occur during an infection of B pseudomallei are not necessarily unique, the
particular virulence and adaptability of the organism allow for an elevated ability to cause harm to the
host.

Biological Warfare Applicability


There are specific factors that contribute to the selection of an organism as a pathogen for weaponization.
In the case of B anthracis, the organism is extremely robust and able to be dispersed in a manner that
allows for ‘area denial’. Also, the organism is non-communnicable meaning there is a reduced likelihood
of unintended infection. For an organism such as Smallpox, the virulence is augmented by the likelihood
that contagion will set in before the first symptoms are apparent leading to epidemic levels if not quickly
identified. For B pseudomallei, the organism is extremely robust in its adaptability to varying
environments as well as mobility. While capable of being inhaled, the organism does not lend itself to
host to host transmission. Indeed, while the afflicted may exhibit flu-like symptoms, the route of infection
often leads away from mucosal surfaces. As an offensive weapon then, area of affect is easily controlled.
More important than the above however is the geographic dispersal of the organism itself. B
pseudomallei is extremely prevalent in locations with little interaction with the developed world. The
ability of a dedicated individual with basic knowledge of microbiology to cultivate B pseudomallei would be
relatively easy. Additionally, the isolation of the organism from the western, developed nations means
that primary care physicians would be unlikely to recognize symptoms that often appear to be something
more common. Finally with the ability to affect not just humans but animals, the threat of cross species
travel means that the organism could be cultivated in a living animal with the intent of introducing the
pathogen to humans further down the line. Despite early experiments with both B pseudomallei and its
close relative B mallei, very little is concretely known about the way to effectively treat and protect against
the threat these organisms present.
Works Cited:
1)Chin, Chui-Yoke, Denise M. Monack, and Sheila Nathan. "Genome wide transcriptome profiling of a
murine acute melioidosis model reveals new insights into how Burkholderia pseudomallei overcomes host
innate immunity." BMC Genomics 11 (2010): 672. Academic OneFile. Web. 2 May. 2011.
http://find.galegroup.com/gps/infomark.do?&contentSet=IAC-
Documents&type=retrieve&tabID=T002&prodId=IPS&docId=A244771236&source=gale&userGroupName
=mlin_s_capecc&version=1.0

2)Detlev Schultze, et al. "Burkholderia pseudomallei misidentified by automated system." Emerging


Infectious Diseases 15.11 (2009): 1799-1801. MEDLINE. EBSCO. Web. 2 May 2011.
http://www.cdc.gov/eid

3)Drexler, Madeline. Emerging Epidemics: The Menace of New Infections. New York: Penguin Books,
2010. Print.

4)Harris, R., & Paxman, J. A Higher Form of Killing. New York: Random House Trade Paperbacks.
August, 2002

5)Larsen, JC, and NH Johnson. "Pathogenesis of Burkholderia pseudomallei and Burkholderia mallei."
Military Medicine 174.6 (2009): 647-651. CINAHL Plus with Full Text. EBSCO. Web. 2 May 2011.

6)“Melioidosis: General Information” Center for Disease Control and Prevention. 18 May 2010. Web. 29
April 2011.
http://www.cdc.gov/nczved/divisions/dfbmd/diseases/melioidosis

7)Ronning, Catherine M., et al. "Genetic and phenotypic diversity in Burkholderia : contributions by
prophage and phage-like elements." BMC Microbiology 10 (2010): 202. Academic OneFile. Web. 2 May.
2011.
http://find.galegroup.com/gps/infomark.do?&contentSet=IAC-
Documents&type=retrieve&tabID=T002&prodId=IPS&docId=A234333419&source=gale&userGroupName
=mlin_s_capecc&version=1.0

8)Tandhavanant, Sarunporn, et al. "Effect of colony morphology variation of Burkholderia pseudomallei


on intracellular survival and resistance to antimicrobial environments in human macrophages in vitro."
BMC Microbiology 10 (2010): 303. Academic OneFile. Web. 2 May. 2011.
http://find.galegroup.com/gps/infomark.do?&contentSet=IAC-
Documents&type=retrieve&tabID=T002&prodId=IPS&docId=A245766507&source=gale&userGroupName
=mlin_s_capecc&version=1.0

9)Zimmerman, Barry E., and David J. Zimmerman. Killer Germs:Microbes and Diseases That Threaten
Humanity. New York: McGraw-Hill, 2002. Print.

10)Wiersinga, Poll, White, Day, and Peacock.“Melioidosis: insights into the pathogenicity of Burkholderia
pseudomallei”. Nature Reviews Microbiology 4, 272-282 (April 2006) | doi:10.1038/nrmicro1385
http://www.nature.com/nrmicro/journal/v4/n4/fig_tab/nrmicro1385_F4.html#figure-title
Multimedia:
Cover Image: http://www.sciencephoto.com/images/download_lo_res.html?id=670063133
Picture of Shiro Ichii: http://en.wikipedia.org/wiki/File:Shiro-ishii.jpg
Anthrax letters: http://www2.fbi.gov/publications/terror/page16.jpg
Burkholderia Pseudomallei Culture:http://www.pathcentre.health.wa.gov.au/images/Pmb21.jpg
Melioidosis symptoms: http://www.nature.com/nrmicro/journal/v4/n4/images/nrmicro1385-f1.jpg
Melioidosis macrophage infection:
http://www.nature.com/nrmicro/journal/v4/n4/fig_tab/nrmicro1385_F4.html#figure-title
Melioidosis infection: http://www.nature.com/nrmicro/journal/v4/n4/fig_tab/nrmicro1385_F5.html

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