BW-ch07.pdf

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

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Melioidosis

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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