CHAPTER ONE INTRODUCTION

REVIEW OF LITERATURE

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2.1 Burkholderia pseudomallei virulence and pathogenesis

2.1.1 Initial epithelial attachment
The primary routes of B.pseudomallei to enter the body through transcutaneous inoculation,
inhalation or aspiration to cause infections. (Currie, Fisher et al. 2000). As a potential way to
cause infection, ingestion has also been studied.(Currie, Mayo et al. 2001). Therefore, both the
eroded skin and the mucosal surface of the epithelial cell layer are initially infected. A thin
polysaccharide film around the bacteria, known as a capsule, helps to arbitrate the attachment of
B.pseudomallei to human pharyngeal epithelial cells.(Ahmed, Enciso et al. 1999). Studies on
attachment inhibition have indicated that B.pseudomallei binds to the aGM1-aGM2 receptor
complex of asialoganglioside. (Gori, Ahmed et al. 1999). The bacterial surface molecule
responsible for binding to aGM1-aGM2 is unknown (Lazar Adler, Govan et al. 2009).
2.1.2 Intracellular invasion
In order to typify the invasion mechanisms of B.pseudomallei, a mutagenesis screen detected a
gene encoding a predicted two-component response regulator (irlRS) that played a role in
epithelial cell invasion, but not macrophages (Jones, DeShazer et al. 1997). Nonetheless, in rats
and C57BL/6 mice, the mutant remained virulent. (Wiersinga, de Vos et al. 2008). This study
indicated that either pseudomallei infection can continue without epithelial cell invasion, or that
the fault of invasion of the observed in vitro tissue culture speaks only of a delay in invasion,
pseudomallei can be noticed initially in vacuoles and later in cytoplasm.(Harley, Dance et al.
1994, Harley, Dance et al. 1998), where the bacteria can begin the process of replication
(Kespichayawattana, Rattanachetkul et al. 2000). Escape from a vacuole is due to the T3SS33
action (Stevens, Wood et al. 2002). T3SS3 mutants describe a vacuolar escape failure that results
in various downstream consequences, including decreased development of actin-tail, intracellular
survival, intracellular spread and cytotoxicity. (Stevens, Wood et al. 2002, Lazar Adler, Govan et
al. 2009). The same pattern of survival, escape, bacterial invasion and replication as inside in

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vitro cell lines was clarified by examining wild-type pseudomallei in the infected mice.
(Gauthier, Hagen et al. 2001).
2.1.3 Survival within macrophages
Without triggering a bactericidal response in the body, Burkholderia pseudomallei will
multiply within phagocytes, including neutrophils, monocytes and macrophages (Jones,
Beveridge et al. 1996, Lazar Adler, Govan et al. 2009). The survival of B.pseudomallei has been
the hot topic of significant study in macrophages, and macrophage cell lines are typically used as
in-vitro model studies for phagocytic cell uptake and survival. Though a degree of lysosome
fusion has been seen inside human macrophages infected with B.pseudomallei, the proliferation
of surviving bacteria engulf the macrophage in the end (Nathan and Puthucheary 2005). Though,
an improved killing of B.pseudomallei occurs if interferon-g stimulates the macrophages.
(Miyagi, Kawakami et al. 1997). Chemical inhibitor research has indicated that the macrophage
killing of B.pseudomallei is largely due to reactive nitrogen intermediates (RNI), while reactive
oxygen intermediates (ROI) play a minor role (Miyagi, Kawakami et al. 1997). Though, in
comparison, knockout mice demonstrated a reduced capacity to clear B.pseudomallei obtained
from ROI deficient, but not RNI-deficient, macrophages. In addition, B.pseudomallei infection
mortality increased in mice with deficient ROI, while mice with deficient RNI were more
resistant. (Breitbach, Klocke et al. 2006). ROI activity therefore appears to be necessary for the
in vivo killing of pseudomallei by macrophages (Lazar Adler, Govan et al. 2009).
2.1.4 Burkholderia pseudomallei evasion of cellular autophagy
The invasion by B.pseudomallei of both epithelial and macrophage cell lines induces
autophagy, a catabolic cellular pathway that forms a component of the innate immune response.
B.pseudomallei is able to deliberately prevent autophagic killing under normal conditions,
however the induction of rapamycin autophagy resulted in a decrease in intracellular bacterial
survival. (Cullinane, Gong et al. 2008). T3SS3 may play a role in autophagy evasion, as a BopA
effector mutant has shown increased autophagic vesicle colocalization and decreased
intracellular survival. (Cullinane, Gong et al. 2008). Thus, B.pseudomallei has developed
mechanisms to suppress innate immune responses from the host, including ROI release and
autophagy stimulation, leading to increased intracellular survival and replication. (Lazar Adler,
Govan et al. 2009).
2.1.5 Cell lysis

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For macrophages, the extent of B.pseudomallei cytotoxicity is strain dependent; some

strains cause macrophage apoptosis, some cause caspase1-dependent cell lysis, whereas others
do not affect the viability of macrophages.(Lazar Adler, Govan et al. 2009). As no cytotoxicity
was observed in an invasion deficient B.pseudomallei T3SS3 mutant or when invasion was
chemically inhibited, bacterial internalization is a prerequisite for macrophage death, (Sun, Lu et
al. 2005). In addition, cell death is at least partly dependent on RpoS because cytotoxicity was
not caused by an rpoS mutant. (Lengwehasatit, Nuchtas et al. 2008). Once 'adequate' bacterial
replication has occurred, macrophage lysis may represent an escape mechanism for
B.pseudomallei.
2.1.6 Intracellular spread
Burkholderia pseudomallei is able to spread intracellularly through membrane protrusions
extending to adjacent cells and through which bacteria move by actin-mediated motility.
(Kespichayawattana, Rattanachetkul et al. 2000, Breitbach, Rottner et al. 2003). The host actin-
associated proteins Arp3, p21 (Arp2/3 complex) and a-actinin complex are recruited by
Burkholderia pseudomallei; however, for actin polymerization, these proteins are not crucial.
(Breitbach, Rottner et al. 2003). BimA, an auto-secreted B.pseudomallei protein, interacts in
vitro with monomeric actin and localizes to the bacterial pole at which actin polymerization takes
place. A mutant of B.pseudomallei bimA was unable to develop actin tails (Stevens, Stevens et
al. 2005); BimA is therefore necessary for intracellular motility mediated by actin. Intracellular
motility based on BimA makes it possible for B.pseudomallei to travel effectively across both
epithelial and macrophage cells while escaping the host immune response. Cell fusion takes
place and multinuclear giant cells (MNGC) are formed as a result of intracellular motility.
(Kespichayawattana, Rattanachetkul et al. 2000). As rpoS mutants fail to stimulate MNGC
formation, this phenotype can be regulated by RpoS. (Utaisincharoen, Arjcharoen et al. 2006).
These data indicate that B.pseudomallei, once adequate bacterial replication has occurred within
an infected cell, stimulates the production of MNGC for intracellular dissemination. Under the
regulation of the B.pseudomallei lfpA gene, MNGC was found to express osteoclast, bone-
remodeling and cell markers; But these osteoclast-like cells are unable to reabsorb the bone
matrix. For virulence, an lfpA mutation was attenuated in both hamsters and BALB/c mice
(Boddey, Day et al. 2007), consistent with the observations that the development of MNGC and
the intracellular spread of pseudomallei are essential for the persistence of infection.

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2.1.7 Secondary spread

Melioidosis is common in localized diseases such as pneumonia and abscesses; however,
B.pseudomallei may spread to secondary locations, including organs such as the liver, spleen or
brain, or to the blood, contributing to septicaemia. (White 2003). The precise mechanism of
secondary spread of B.pseudomallei is unknown, though macrophage invasion might permit the
spleen and other organs to be transported through the lymphatic system. Foci containing
Burkholderia pseudomallei are typically found in the spleens of chronically infected mice.
(Lazar Adler, Govan et al. 2009). In addition, inflammation at the site of the infection allows
greater access to the circulatory system; due to the defensive effects of the polysaccharide
capsule and lipopolysaccharide, pseudomallei can live inside the human serum (Reckseidler-
Zenteno, DeVinney et al. 2005). Resistance to complement-mediated killing is mediated by both
capsules and lipopolysaccharides, whereas lipopolysaccharide is also responsible for resistance
to cationic peptides. (Lazar Adler, Govan et al. 2009).
2.1.8 Latency
Patients recovering from B.pseudomallei septicaemia have retained elevated levels of
antibodies for years, indicating either constant exposure to the body or intracellular or cryptic
site sequestration of bacteria. (Vasu, Vadivelu et al. 2003). Neither the latency site(s) nor the
processes by which B.pseudomallei remains undetected are apparent. (Gan 2005). Recurrent
disease, however, is prevalent, occurring in 6-13 percent of cases, mostly due to relapse rather
than reinfection, especially when primary infection occurs within a year. (Maharjan, Chantratita
et al. 2005). The bacteria may remain latent for extended periods before trauma or
immunosuppression has been activated; cases of latency of 18, 26 and 62 years have been
recorded. (Lazar Adler, Govan et al. 2009). Such a long latency periods indicate that
B.pseudomallei can enter a dormant state in which immune surveillance can be prevented, most
likely in an intracellular area. (Lazar Adler, Govan et al. 2009).
2.2 The role of the host response in the molecular pathogenesis of B. pseudomallei
Although healthy people can contract melioidosis, most patients have an inherent
predisposition, meaning that the patient's immunological condition influences the initiation and
development of the illness. In general, diabetes mellitus and renal failure in melioidosis patients
are common underlying conditions; other variables that contribute to immune suppression have
also been identified as risk factors, such as alcohol abuse. (White 2003). There are many disease

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outcomes (asymptomatic, acute, chronic or latent) in melioidosis that are thought to be

determined by the host immune response. (Gan 2005). Acute (BALB/c) and chronic (C57BL/6)
infection models of murine melioidosis resemble acute and chronic diseases in humans. (Lazar
Adler, Govan et al. 2009). A substantially stronger innate immune response characterizes the
acute melioidosis found in BALB/c mice. (Lazar Adler, Govan et al. 2009). Hyperproduction of
proinflammatory cytokines, however, results in an ineffective cellular response that does not
control the infection and leads to tissue damage and multiple organ failure. The chronic infection
observed in C57BL/6 mice, on the other hand, shows a modest increase in cytokines that allows
mice to temporarily contain B.pseudomallei within phagocytes, giving time for an adaptive
immune response to happen.(Lazar Adler, Govan et al. 2009). However, to date, the relative
importance of both the innate and the adaptive immune responses of the cell-mediated and
humoral arms remains uncertain. (Cheng and Currie 2005, Gan 2005).
2.2.1 Interaction of B. pseudomallei with the innate immune response
The alternative complement pathway is activated by Burkholderia pseudomallei, but the
membrane attack complex is retained on an external polysaccharide and is therefore not
bactericidal. (Lazar Adler, Govan et al. 2009). Opsonization with a complement improves, but is
not sufficient for, phagocyte absorption and does not necessarily contribute to intracellular
killing of the bacteria. (Kespichayawattana, Rattanachetkul et al. 2000, Lazar Adler, Govan et al.
2009). Resistance to lysosomal defensins and cationic peptides has been shown by
B.pseudomallei. (Gan 2005). Attributed to the presence of the capsule and lipopolysaccharide,
these resistance mechanisms allow B.pseudomallei to live inside phagocytes and in human
serum. Mouse tissue shows a quick influx and activation of neutrophils following infection with
B.pseudomallei. (Barnes, Ulett et al. 2001). The acute B.pseudomallei infection is identified
when C57BL/6 mice are deprived of neutrophils. (Easton, Haque et al. 2007), showing the
significance of neutrophils to innate immunity. Macrophages are also, however, important for the
control of infection with B.pseudomallei. Macrophage loss from BALB/c or C57BL/6 mice
greatly increases death rates. (Breitbach, Klocke et al. 2006, Barnes, Williams et al. 2008). In
BALB/c mice, Burkholderia pseudomallei infection does not attract macrophages to the same
degree as in C57BL/6 mice. Macrophages recruited by C57BL/6 mice were suggested to
temporarily produce B.pseudomallei, contributing to chronic melioidosis seen in these animals.
(Barnes, Ulett et al. 2001). Compared with healthy people, macrophages from melioidosis

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patients display a decreased degree of lysosomal fusion, leading to increased bacterial numbers.
(Puthucheary and Nathan 2006). These data indicate that an inadequate cellular innate immune
response occurs in acute melioidosis. Toll-like receptors (TLRs) identify and trigger an
inflammatory immune reaction to retained pathogen-associated molecular patterns. Using
various signaling adaptor proteins, including MyD88 and TRIF, induction of TLRs happens. As
a result of decreased neutrophil recruiting and activation, MyD88 knockout mice showed
increased susceptibility to B.pseudomallei infection. (Wiersinga, Wieland et al. 2008). The
expression of TLR1, TLR2 and TLR4 and its coreceptor CD14 increased in melioidosis patients
with septic shock; the expression of TLR2, TLR4 and CD14 lowered after recovery. (Wiersinga,
Wieland et al. 2007). In epithelial reporter cell lines, Burkholderia pseudomallei triggers TLR2,
TLR4 and TLR5 receptors and induces IL-8 secretion via NF-kBB (Hii, Sun et al. 2008). The
association between B.pseudomallei and TLR2, TLR4 and CD14 was confirmed by decreased
tumor necrosis factor (TNF) expression in knockout mice leukocytes. TLR4 knockout mice
showed wild-type mortality following B.pseudomallei infection, while both TLR2 and CD14
knockout mice showed decreased death, bacterial burdens and inflammation, as evaluated
clinically and by cytokine level measurement. Purified lipopolysaccharide B.pseudomallei was
discovered to trigger via TLR2 (Wiersinga, Wieland et al. 2007, Wiersinga, de Vos et al. 2008).
These data therefore indicate that proinflammatory cytokine release induced by B.pseudomallei
lipopolysaccharide leads to infection severity and results in acute melioidosis.

2.2.2 Interaction of B. pseudomallei with the humoral immune response

B.pseudomallei antibodies are typical for individuals residing within melioidosis endemic
regions, although the percentages of seropositive individuals differ greatly across regions and
subpopulations. (Barnes, Warner et al. 2004, Lazar Adler, Govan et al. 2009). This variance may
be due to the cross-reaction of B.pseudomallei antigens with Burkholderiaceaee species of
similar avirulence (Cheng and Currie 2005, Gilmore, Barnes et al. 2007). The function of
antibodies is inconclusive in protecting against infections. In melioidosis patients, an antibody
screen identified anti-lipopolysaccharide antibodies as defensive (Lazar Adler, Govan et al.
2009). However, despite showing that these antibodies induced phagocyte killing in vitro, a
scientist Ho in 1997 found no association between the magnitude or survival of the disease and

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capsule or lipopolysaccharide antibodies. In particular, persistent infections may happen in the

presence of high levels of antibodies (Vasu, Vadivelu et al. 2003).
2.2.3 Interaction of B. pseudomallei with the cellular immune response
A cell-mediated immune (CMI) response, in which T cells play an important role, is
needed for bacterial evacuation once intracellular invasion by B.pseudomallei has occurred.
Melioidosis patients, though, exhibit reduced counts of T-cells (Ramsay, Ketheesan et al. 2002).
Following B.pseudomallei lysate stimulation, T cells from subclinical melioidosis patients
displayed higher levels of proliferation and higher development of IFN-g than those from clinical
melioidosis patients. These data indicate that for defense against the progression of
B.pseudomallei infection, a strong CMI response is necessary (Barnes, Warner et al. 2004) in
BALB/c and C57BL/6 mice after immunization with B.pseudomallei, CMI responses are shown.
This CMI response was transferred by lymphocyte transfer to naive mice, but no defense was
seen against a subsequent challenge (Barnes and Ketheesan 2007).  Reduction of CD4 T cells,
but not of CD8 T cells, from BALB/c B.pseudomallei immunized mice led to an increase
susceptibility to infection. (Haque, Chu et al. 2006). For B-cell isotype switching and activation
of CD8 cells, but also for phagocyte activation, CD4 T cells are extremely important; therefore,
these data indicate that a comprehensive cellular response is needed to regulate B.pseudomallei
infection. In addition, only when both lymphocytes and macrophages were present was
maximum bactericidal activity observed against B.pseudomallei (Lazar Adler, Govan et al.
2009). In Taylor outbred mice, whose cellular response included an initial neutrophil infiltration
accompanied by macrophage and lymphocyte influx, a sublethal, chronic infection, indicating
containment of pseudomallei infection, was observed. (Lazar Adler, Govan et al. 2009).
2.2.4 Cytokines play an important role in regulating the immune response to B.
pseudomallei infection.
However, these regulatory mechanisms fail in acute illness, resulting in an increased
inflammation, (Gan 2005). High levels of proinflammatory cytokines (IL-6, IL-12, IL-15, IL-18,
TNF-a and IFN-g) are developed in patients with acute melioidosis; serum levels of some of
these have been shown to be substantially higher in cases of fatal melioidosis. (Lazar Adler,
Govan et al. 2009). A high serum level of either IL-6 or IL-18 is also known an indicator of
mortality. Thus the, the host's immune reaction makes a substantial contribution to melioidosis
pathogenesis. A clear image of the cytokine responses to B.pseudomallei has been given using

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the murine model of melioidosis. In the acute melioidosis BALB/c mouse model, increased
levels of proinflammatory cytokines are reported, while slightly increased levels of
proinflammatory cytokines are noticed in the C57BL/6 chronic melioidosis mouse model. (Lazar
Adler, Govan et al. 2009). In addition, examination of the kinetics of cytokine expression
revealed that cytokines peaked in BALB/c mice earlier (24-48 h) than those in C57BL/6 mice
(48-72 h). Instead of the B.pseudomallei strain's virulence, cytokine levels tend to correlate with
bacterial amounts. The high levels of cytokine observed in BALB/c mice are therefore a direct
product of the higher bacterial burdens observed in these animals. (Lazar Adler, Govan et al.
2009).
In the melioidosis mouse model, IFN-g has been said to be important for an innate immune
response against B.pseudomallei. IFN-g development is stimulated by Burkholderia
pseudomallei, which then stimulates T cells and natural killer (NK) cells, reinforcing the CMI
response (Lauw, Simpson et al. 2000). Due to increased bacterial loads, administration of anti-
IFN-g antibodies resulted in acute melioidosis in C57BL/6 mice at usually sublethal doses.
(Breitbach, Klocke et al. 2006, Easton, Haque et al. 2007). IFN-g knockout mice were highly
vulnerable to infection with B.pseudomallei, whereas mice with lymphocyte deficiency had
moderate resilience, demonstrating the value of IFN-g derived from NK cells. Within 5 h of
B.pseudomallei infection, NK cells are detectable at the infection site and produce 60-80 percent
of the secreted IFN-g (Easton, Haque et al. 2007, Lazar Adler, Govan et al. 2009). Compared to
C57BL/6 mice, IFN-g hyperproduction in BALB/c mice may be due to differences in receptors
found on T cells and NK cells in BALB/c mice. (Koo and Gan 2006). The administration of anti-
TNF-a antibodies also led to an increased sensitivity to infection with B.pseudomallei, but not at
the same degree as anti-IFN-g antibodies. Knockout mice also showed an increased vulnerability
to B.pseudomallei infection for either TNF-a or its receptors; these mice showed increased
inflammatory influx based on neutrophils and linked necrosis. As a result of the interaction of
B.pseudomallei with the macrophage cell surface, TNF-a, mainly formed by macrophages, was
found to be expressed. (Lazar Adler, Govan et al. 2009)
2.3 B. pseudomallei eradication therapy
2.3.1 Clinical Management
While it is very difficult to make a diagnosis on clinical grounds alone, observational care
should be begun for patients from endemic areas with indicative clinical and epidemiological

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characteristics (presence of risk factors and occupational or seasonal exposure). It has been
shown that a lack of clinical suspicion result in delayed anti-melioidosis treatment leads to
mortality (although statistically non-significant). Some studies indicate that about a third of
patients have received suitable melioidosis antibiotics upon admission (Tang, Lim et al. 2019).
Further evaluations and reassessments of diagnosis and care should be considered if patients do
not improve after 3 to 7 days of adequate treatment for melioidosis and culture outcomes remain
negative. Burkholderia pseudomallei is vulnerable to the bacteriostatic agents doxycycline,
chloramphenicol and trimethoprim-sulfamethoxazole, and is almost always susceptible to β-
lactam antibiotics such as ceftazidime, meropenem, imipenem and coamoxiclav (with varying
bactericidal activity). It is, however, immune to penicillin, ampicillin, and cephalosporins of the
first and second generations, gentamicin, tobramycin, streptomycin, macrolides and polymyxins
(Lipsitz, Garges et al. 2012, Crowe, McMahon et al. 2014). Clonal groups of gentamicin
sensitive isolates typically found in Sarawak, Malaysia, are a caveat to this (Podin, Sarovich et
al. 2014) As 10.9 percent of isolates from Hanoi (Vietnam) were immune to trimethoprim-
sulfamethoxazole by the E-test process, caution should also be provided for continuous
monitoring of antimicrobial susceptibility (including shifting minimum inhibitory concentration
values) (Nhung, Van et al. 2019). In addition, during therapy, B.pseudomallei has the potential to
develop decreased tolerance to meropenem (cases becoming refractory amid aggressive therapy),
triggered by multiple mutations and overexpression of efflux pumps for multidrug resistance-
nodulation-division (Sarovich, Webb et al. 2018). In practice, carriage of the blaOXA-57 (class
D β-lactamase) gene has been identified, but phenotypically isolates remain susceptible to
imipenem. (Amladi, Devanga Ragupathi et al. 2019) In vitro evidence has been shown for the
use of novel agents such as ceftolozane-tazobactam in the management of melioidosis (Slack,
Parsonson et al. 2018) but, the function of ceftazidim has not yet been replaced by any. After a
2010 expert workshop that revised previous consensus-based guidelines, standardized guidelines
for melioidosis therapy have been published by the U.S. CDC (Lipsitz, Garges et al. 2012). In
addition, the so-called Darwin guideline for the period of oral and intravenous phases of therapy
was released in Seminars in 2015, based on clinical presentation and dosing guidelines for
patients with srenal disability (Currie 2015). Antibacterial therapy is split into the initial intensive
phase and the eventual eradication phase
2.3.2 Acute Intensive

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Intravenous ceftazidime or meropenem is the preferred choice of treatment. Observational

evidence, however, support the recommendation of using meropenem as the drug of choice in
septic shock melioidosis. Initial intensive therapy should last a minimum of 10 to 14 days.
Longer durations are needed for critically ill patients. The Darwin cohort had an average period
of approximately 4 weeks of intensive therapy, resulting in a 1.2 percent relapse rate. The
median time for fever resolution is up to 9 days (Virk, Mukhopadhyay et al. 2020). Studies have
not shown any survival gain, although some doctors recommend combination therapy with
trimethoprim-sulfamethoxazole (Chierakul, Anunnatsiri et al. 2005, Chierakul, Anunnatsiri et al.
2007)
2.3.3 Eradication Treatment
To avoid recrudescence of the patient's disease or relapse, step eradication therapy with oral
antibiotics is recommended. The preferred agent for eradication therapy is trimethoprim-
sulfamethoxazole, followed by co-amoxiclav or doxycycline as the second alternative (Lipsitz,
Garges et al. 2012). Adverse effects in up to 40 percent of patients are recorded due to the
necessary high dose and long-term course of trimethoprim-sulfamethoxazole (Chetchotisakd,
Chierakul et al. 2014). Doctors should be mindful that dosing with co-amoxiclav can be
troublesome, and if co-amoxiclav or doxycycline is used, acquired resistance is well recorded
(Lipsitz, Garges et al. 2012). Eradication therapy failure is related to poor adherence, more
serious multifocal infections, and fewer than 8 weeks of eradication therapy period. The low rate
observed in Australia is due to the prolonged intensive initial treatment (Pitman, Luck et al.
2015).

2.3.4 The Search for Adjunctive

For single, large abscesses or extensive osteomyelitis/septic arthritis, surgical drainage or
pleurodesis is generally required. Mycotic aneurysms are a medical emergency, usually
involving the injection of surgical material, an indicator of trimethoprim-sulfamethoxazole
lifelong suppressive treatment. Many other novel therapeutics, including new antibiotics,
antibiotic potentiation, antibody therapy, antimicrobial peptides, and immune modulation, have
also been developed and tested in in vitro models (Tapia, Sanchez-Villamil et al. 2019) However,
in human patients with melioidosis and extreme sepsis, the granulocyte-colony stimulating factor

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(lenograstim) has been studied to potentially resolve functional neutrophil defects seen in
patients with melioidosis. Despite promising observational results, unfortunately, (Cheng,
Stephens et al. 2004) While patients lived longer, a randomized-controlled study failed to
demonstrate a survival advantage. State-of-the-art intensive care treatment dramatically reduces
death in patients with melioidosis and sepsis/septic shock, so critical care infrastructure
expansion is paramount, especially in LMIC. Recrudescence has decreased from 10% of cases to
<5% with clinical changes. New infections are now more frequent than recrudescence in
melioidosis survivors.(Virk, Mukhopadhyay et al. 2020)
2.3.5 Vaccines
No vaccines against melioidosis are commercially available for human use. While some
vaccine candidates in murine infection models provide partial defense against melioidosis, some
have been tested in non-human primates or humans. Antibody responses, however, play a key
role in protecting against melioidosis and the response of CD8Ş T cells is associated with higher
survival (Virk, Mukhopadhyay et al. 2020). To achieve maximum safety, a multivalent vaccine
containing various immunogenic bacterial components is likely to be required. A subunit vaccine
using purified 6-deoxyheptan CPS from pseudomallei linked to the recombinant CRM197
diphtheria toxin mutant, together with purified pseudomallei Hcp1 (T6SS-1 effector) and TssM
(deubiquitinase) proteins has been developed for this purpose. High-titer immunoglobulin G
(IgG) and opsonizing antibody responses result from immunization of C57BL/6 mice with
CPSCRM197, while Hcp1 and TssM develop high-titer IgG and robust gamma interferon-
secreting T cell responses. A mouse model of all this combination of vaccines showed that 100%
of mice sustained a lethal inhalation challenge, with up to 70% exhibiting a sterilizing immune
response (Burtnick, Shaffer et al. 2018). Reassuringly, these results of a subunit vaccine
generated by Paul Brett and Mary Burtnick at the University of Nevada, Reno, contributed to the
first phase 1 clinical trial to launch at Oxford University, United Kingdom in 2021 in volunteer
subjects with and without diabetes (Prof. S. Dunachie, personal communication). It is also worth
noting that other candidates for conjugated vaccines also demonstrate potential, and Morici et al.
have recently published a comprehensive analysis. Modeling has also shown that a melioidosis
vaccine targeting high-risk populations in susceptible areas can be beneficial and cost-effective
by reducing the burden of illness. (Virk, Mukhopadhyay et al. 2020).

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CHAPTER ONE INTRODUCTION