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Melioidosis
Chapter 7
MELIOIDOSIS
NICHOLAS J. VIETRI, MD * ; and DAVID DESHAZER, P h D
INTRODUCTION
INFECTIOUS AGENT
MILITARY RELEVANCE
DISEASE
Epidemiology
Pathogenesis
Clinical Disease
Diagnosis
Treatment
Prevention
SUMMARY
* Major, Medical Corps, US Army; Infectious Diseases Physician and Principal Investigator, Division of Bacteriology, US Army Medical Research
Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702; formerly, Infectious Diseases Fellow, Department of Medicine,
BrookeArmyMedicalCenter,SanAntonio,Texas
Microbiologist,DivisionofBacteriology,USArmyMedicalResearchInstituteofInfectiousDiseases,1425PorterStreet,FortDetrick,Maryland21702;
formerly,Microbiologist,PostdoctoralFellow,DepartmentofMicrobiologyandInfectiousDiseases,UniversityofCalgary,Calgary,Alberta,Canada
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MedicalAspectsofBiologicalWarfare
INTRODUCTION
In 1911 Captain Alfred Whitmore and Dr CS Krish-
naswami described a previously unrecognized disease
that was prevalent among the ill-nourished and ne-
glected inhabitants of Rangoon, Burma. 1 The new dis-
ease resembled glanders, a zoonotic disease of equines. 2
Whitmore and Krishnaswami isolated a gram-negative
bacillus that resembled the glanders bacillus, Bacillus
mallei , from postmortem tissue samples. 3 However, the
new bacillus could be differentiated from Bmallei by its
motility, luxuriant growth on peptone agar, and wrin-
kled colony morphology; it was subsequently named
Bacilluspseudomallei . 3,4 Whitmore’s detailed account of
the first 38 human cases of this disease demonstrated
that most of those affected were morphine injectors who
died of septicemia with abscesses in multiple organs. 4
As a result, the disease became known as “Whitmore’s
disease” or “morphine injector’s septicemia.” 5,6 In 1921
Stanton and Fletcher reported an outbreak of a septi-
cemic disease in a guinea pig colony at the Institute for
Medical Research in Kuala Lumpur. 7 Stanton and Fletch-
er isolated an infectious agent from diseased animals
that was indistinguishable from Whitmore’s bacillus,
and they named it “melioidosis” (a Greek term meaning
glanders-like illness) to describe this new disease of the
tropics. 7 Stanton and Fletcher subsequently published
a classic monograph in 1932 describing their observa-
tions of melioidosis in humans and animals occurring
in Burma, Malaya, French Indochina, and Ceylon. 8
Melioidosis is regarded as an emerging infectious
disease and a potential bioterrorism threat. 9-11 The
etiologic agent of melioidosis is present in water and
soil in tropical and subtropical regions; it is spread to
humans through direct contact with the contaminated
source. Clinical manifestations range from subclinical
infection to overwhelming septicemia that resembles
disseminated or localized, suppurative infection at-
tributable to a variety of pathogens, resulting in the
nickname “the remarkable imitator.” 12 The majority of
melioidosis cases have identified risk factors, includ-
ing diabetes, alcoholism, chronic renal disease, cystic
fibrosis, and steroid abuse. 13 AIDS does not seem to be
a major risk factor for melioidosis. Healthy individuals
can also contract melioidosis, especially if they work in
muddy soil without good hand and foot protection. 14
Many animal species are susceptible to melioidosis,
including sheep, goats, horses, swine, cattle, dogs, and
cats. 15 Numerous review articles on melioidosis have
been published since 1990. 11,13-27
INFECTIOUS AGENT
The bacterium that causes melioidosis, now des-
ignated Burkholderia pseudomallei , 28 has undergone
numerous name changes since its original classification
as Bpseudomallei , including ( a ) Bacteriumwhitmori , ( b )
Bacilluswhitmori , ( c ) Pfeifferellawhitmori , ( d ) Pfeifferella
pseudomallei , ( e ) Actinobacilluspseudomallei , ( f ) Lofflerella
whitmori , ( g ) Flavobacteriumpseudomallei , ( h ) Malleomy-
cespseudomallei , and ( i ) Pseudomonaspseudomallei . The
nonsporulating, gram-negative bacillus is an environ-
mental saprophyte found in surface waters and wet
soils in endemic regions. 29-36 Individual cells, which are
approximately 0.8 x 1.5 μm, have a polar tuft of two to
four flagella and exhibit bipolar staining with a “safety
pin” appearance. 37,38 Bpseudomallei is metabolically ver-
satile and can grow on numerous carbon sources. 28,39
Anaerobic growth is possible, but only in the presence
of nitrate or arginine. 11 The microbe accumulates intra-
cellular stores of poly-β-hydroxybutyric acid and can
survive in distilled water for years. 10,40,41 The optimal
survival temperature for B pseudomallei is between
24°C and 32°C, but it can grow at temperatures up
to 42°C. 42,43 Bpseudomallei demonstrates considerable
interstrain and medium-dependent colony morphol-
ogy. 44-46 The oxidase-positive organism can grow on a
variety of microbial media, but Ashdown’s selective
medium is often used for isolating Bpseudomallei from
environmental and clinical specimens. 47 Two distinct
colony phenotypes are commonly observed on this
medium (Figure 7-1), probably because of the differ-
ential uptake of crystal violet and neutral red or the
differential production of ammonia and oxalic acid. 47,48
Most strains appear lavender after 2 to 3 days of incu-
bation at 37°C, but some isolates appear deep purple
(see Figure 7-1). After 5 days at 37°C, the colonies often
become dull and wrinkled (see Figure 7-1) and emit a
distinctive sweet earthy smell. Other selective media
have also been used to isolate B pseudomallei from
contaminated specimens. 49,50
The complete genome sequence of B pseudomallei
K96243, a strain isolated in 1996 from a 34-year-old
diabetic patient in Khon Kaen, Thailand, was recently
determined. 51 The 7.25-megabase pair (Mb) genome
was composed of two circular replicons, termed chro-
mosome 1 (4.07 Mb) and chromosome 2 (3.17 Mb). The
G + C content of the genome was 68% and predicted
to encode 5,855 proteins. Chromosome 1 encoded a
high proportion of core housekeeping functions (DNA
replication, transcription, translation, amino acid and
nucleotide metabolism, basic carbohydrate metabolism,
and cofactor synthesis); and chromosome 2 encoded a
high proportion of accessory functions (adaptation to
atypical conditions, osmotic protection, and secondary
148
Melioidosis
a
b
Fig. 7-1. Burkholderia pseudomallei colony morphologies as
demonstrated on Ashdown’s selective medium supple-
mented with 100 μg/mL streptomycin. Plates were incubated
for 3 days at 37°C ( a ) and 5 days at 37°C ( b ).
Photographs: Courtesy of David Deshazer, PhD, US Army
Medical Research Institute of Infectious Diseases, Fort De-
trick, Maryland.
metabolism). 51 Plasmid-like replication genes and ac-
cessory genes on chromosome 2 suggest it may have
been derived from a plasmid (or megaplasmid) that
became an indispensable replicon by acquiring essential
functions such as tRNA genes, amino acid biosynthesis
genes, and energy metabolism genes. There are 16 “ge-
nomic islands” in the Bpseudomallei K96243 genome that
appear to have been acquired through horizontal gene
transfer. 51 Mobile genetic elements, such as prophages,
insertion sequences, and integrated plasmids, account
for most of the laterally acquired genomic sequences.
Recent studies have shown that Bpseudomallei strains
exhibit significant genomic diversity and that much of
the genetic heterogeneity is caused by laterally acquired
mobile genetic elements. 51-56 These genomic islands may
provide strains that harbor a metabolic and/or virulence
advantage over strains that do not contain such se-
quences. Similarly, autonomously replicating plasmids
are variably present in Bpseudomallei isolates, but little is
known about their biological significance. 27,57-59 Recently,
the draft genome sequences of an additional nine B
pseudomallei isolates (1710a, 1710b, 406e, 1106a, 1106b,
S13, Pasteur 52237, 668, and 1655) were determined
and deposited in Genbank, dramatically enhancing the
amount and diversity of genome sequence data avail-
able for the study of Bpseudomallei .
MILITARY RELEVANCE
Throughout the 20th century, melioidosis had an
impact on the health of soldiers serving in Asia dur-
ing times of war and peace. 60 Sporadic melioidosis
infections occurred in US and Japanese soldiers dur-
ing World War II, 38,61,62 and recrudescent melioidosis
cases in World War II veterans were also reported. 63,64
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MedicalAspectsofBiologicalWarfare
During the French Indochina War (1946–1954), there
were at least 100 melioidosis cases among French forces
during their fight against the resistance movement
led by the Viet Minh. 19,60 Fewer than 300 melioidosis
cases occurred among US soldiers during the Vietnam
War, 19 and additional cases did not surface until years
after the war’s end, leading to the nickname “Vietnam
Time Bomb.” 65-67 Twenty-three melioidosis cases were
reported in the Singapore armed forces from 1987 to
1994. 68 The infection rate in these relatively healthy
servicemen was approximately 4-fold higher than the
rate in Singapore’s general population, suggesting that
close contact with the soil during military training may
lead to an increased risk for melioidosis.
Bpseudomallei is a Centers for Disease Control and
Prevention Category B biological terrorism agent that
must be handled in biosafety level 3 laboratories. 9
Biosafety level 3 facilities incorporate specialized
negative-air pressure ventilation systems, well-defined
biosafety containment equipment, and protocols to
study agents that can be transmitted through the air
and cause potentially lethal infection. Category B
agents have the potential for large-scale dissemination
with resultant illness and death, but generally would
be expected to have lower medical and public health
impact than Category A agents. 9 B pseudomallei was
studied by the United States, the former Soviet Union,
and possibly Egypt as a potential biological warfare
agent, but was never used in this capacity. 69-71 However,
Bmallei was used as a biological warfare agent during
the American Civil War, World War I, World War II, and
in Afghanistan between 1982 and 1984. 2,70,72,73 The use-
fulness of Bpseudomallei as a biological warfare agent
is unknown, but the ease of acquiring strains from the
environment, the ability to genetically manipulate the
agent to be multiply antibiotic resistant, and the lack
of a melioidosis vaccine make this possibility a seri-
ous concern.
DISEASE
Epidemiology
exhibit intermediate susceptibility to experimental
infection with Bpseudomallei , but the LD 50 for mice var-
ies widely depending on the route of infection, mouse
strain, and bacterial strain. 80,81,84,88
Basic research on this pathogen has progressed
rapidly over the past 5 years because of fears that B
pseudomallei may be used as a biological weapon. The
identification of virulence factors has been facilitated
by the availability of genomic sequence data 51 and the
existence of a nonpathogenic B pseudomallei -like spe-
cies designated Bthailandensis . 89-91 Bpseudomallei and B
thailandensis strains are genetically and immunologically
similar to one another, but Bthailandensis is avirulent in
animal models of infection and rarely causes disease in
humans. Genetic determinants that confer enhanced
virulence in Bpseudomallei relative to Bthailandensis have
been identified by comparative analysis of genomic
DNA from these species. 53,92,93 Exhibit 7-1 provides a
brief description of all known Bpseudomallei virulence
factors, their mechanisms of action, and their relative
importance in animal models of melioidosis.
B pseudomallei is a facultative intracellular patho-
gen that can replicate and survive in phagocytic and
nonphagocytic cell lines. 94-99 After the initial phase
of infection, researchers postulate that Bpseudomallei
can persist in a dormant stage in macrophages for
months or years. 99 Melioidosis has the potential for a
long latency period, and Bpseudomallei’s intracellular
persistence could provide a mechanism by which this
occurs. Intracellular survival and cell-to-cell spread
may also provide B pseudomallei protection from the
humoral immune response.
Melioidosis cases have been increasingly reported
from countries located between 20°N and 20°S in
latitude, with the greatest concentration in Vietnam,
Cambodia, Laos, Thailand, Malaysia, Singapore, and
northern Australia. 11,13,20 Melioidosis has also been
observed in the South Pacific, Africa, India, and the
Middle East. In addition, sporadic melioidosis cases
have occurred in the Western Hemisphere in Aruba,
Brazil, Mexico, Panama, Ecuador, Haiti, Peru, and
Guyana. 11,13,20 In endemic regions, the disease occurs
in humans, sheep, goats, horses, swine, cattle, dogs,
cats, and other animals. 15,24 Melioidosis cases that occur
in temperate regions often result from recent travel to
endemic areas. 18,74-77
Pathogenesis
Several animal models of melioidosis have been
developed to study pathogenesis, virulence factors,
and efficacy of antibiotics and vaccines. 78-86 In gen-
eral, hamsters and ferrets are highly susceptible to
experimental melioidosis (median lethal dose [LD 50]
of < 10 2 bacteria), and rats, pigs, and rhesus monkeys
are relatively resistant (LD 50 of > 10 6 bacteria). Infant
rats can be made more susceptible to infection by in-
traperitoneal injection of streptozotocin, a compound
that induces diabetes. 82,87 The LD 50 of Bpseudomallei for
nondiabetic infant rats is greater than 10 8 bacteria in
streptozotocin-induced diabetic infant rats; the LD 50
is approximately 10 4 bacteria. Mice and guinea pigs
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Melioidosis
EXHIBIT 7–1
CANDIDATE VIRULENCE FACTORS OF BURKHOLDERIA PSEUDOMALLEI
Factor
Description
Capsule
A 200-kd group 3 capsular polysaccharide composed of a homopolymer of -3)-2- O -acetyl-
6-deoxy-ß-D- manno -heptopyranose-(1-. 1 Capsule mutants are highly attenuated in hamsters
and mice. 2,3 The capsule may contribute to survival in serum by reducing complement factor
C3b deposition. 4
TTSS
Bpseudomallei harbors three distinct TTSS loci: (1) TTSS1, (2) TTSS2, and (3) TTSS3. 5 The TTSS1
and TTSS2 loci are similar to TTSS genes of the plant pathogen Ralstoniasolanacearum and are
not necessary for virulence in hamsters. 5 The TTSS3 locus is similar to the TTSS in Salmonella
and Shigella 6 and is required for full virulence of Bpseudomallei in both hamsters and mice. 5,7 The
effector proteins of TTSS3 facilitate the invasion of epithelial cells and escape from endocytic
vesicles. 6,8
Quorum sensing
Bpseudomallei encodes three luxI homologues that produce at least three quorum-sensing mol-
ecules: (1) N-octanoyl-homoserine lactone (C8-HSL), 9,10 (2) N-decanoyl-homoserine lactone (C10-
HSL), 9,11 and (3) N-(3-hydroxyoctanoyl)-L-homoserine lactone (3-hydroxy-C8-HSL). 9 It also has
five luxR homologues to sense these signals. Mutations in all of the luxI and luxR homologues
result in strains with decreased virulence in hamsters and mice, 9,11 but the virulence-associated
genes regulated by this complex quorum-sensing system are under investigation.
LPS O-antigen
An unbranched heteropolymer with repeating D-glucose and L-talose units with the structure -3)-
ß-D-glucopyranose-(1–3)-6-deoxy-a-L-talopyranose-(1-. 12-14 LPS O-antigen mutants are attenuated
in hamsters, guinea pigs, and infant diabetic rats and are killed by serum. 15 This factor promotes
survival in serum by preventing killing by the alternative pathway of complement. Levels of anti-
LPS O-antigen antibodies are significantly higher in patients who survive than in those who die. 16
Flagellin
A surface-associated 43-kd protein that is required for motility. 17,18 Flagellin mutants are attenuated
in mice, 19 but not in hamsters or infant diabetic rats. 18 Passive exposure studies demonstrated
that flagellin-specific antiserum was capable of protecting infant diabetic rats from challenge
with Bpseudomallei . 17
Type II secretion
Required for the secretion of several exoproducts, including protease, lipase, and phospholi-
pase C. 20 The products secreted by this pathway appear to play a minor role in Bpseudomallei
pathogenesis. 21
Type IV pilin
Bpseudomallei K96243 encodes four complete type IV pilin clusters. 22 A mutation in pilA , a gene
encoding a type IVA pilin subunit, resulted in a strain exhibiting decreased attachment to cul-
tured respiratory cell lines relative to wild-type. The pilA mutant was not attenuated in mice
by the intraperitoneal challenge route, but was slightly attenuated by the intranasal challenge
route. 23
Biofilm formation
The extracellular slime matrix produced by Bpseudomallei appears to be polysaccharide in nature,
but the exact structure is unknown. 24 Biofilm mutants were not attenuated in the mouse model
of melioidosis, suggesting that the biofilm plays a relatively minor role, if any, in virulence. 24
Malleobactin
A water-soluble siderophore of the hydroxamate class. 25 The compound is capable of scav-
enging iron from both lactoferrin and transferrin in vitro. 26 The genes encoding malleobactin
biosynthesis and transport were recently identified, but malleobactin mutants were not tested
in animal models of melioidosis. 27
Rhamnolipid
A 762-Da glycolipid with the structure 2-O-a -L-rhamnopyranosyl-a -L-rhamnopyranosyl-ß-
hydroxytetradec anoyl-ß-hydroxytetradecanoate (Rha-Rha-C14-C14). 28 Rhamnolipid-treated
cell lines exhibit profound morphological alterations, but the role of this glycolipid in virulence
remains unknown. 29
EPS
A linear unbranched polymer of repeating tetrasaccharide units composed of D-galactose and
3-deoxy-D- manno -octulosonicacid (KDO),with the following structure: -3)-2- O -Ac-ß-D-Gal p -(1-
4)-a -D-Gal p -(1-3)-ß-D-Gal p -(1-5)-ß-D-KDO p -(2-. 30-32 EPS is not produced by the closely related
nonpathogenic species Bthailandensis , suggesting that it may be a virulence determinant of B
pseudomallei . EPS is probably produced during infection because sera from melioidosis patients
contain IgG and IgM antibodies to EPS. 31,33
Endotoxin
The lipid A portion of Bpseudomallei LPS contains amide-linked 3-hydroxyhexadecanoic acids,
( Exhibit 7-1 continues )
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