Lipopolysaccharide from Burkholderia thailandensis E264 provides protection in a murine model of melioidosis.
ABSTRACT Burkholderia thailandensis is a less virulent close relative of Burkholderia pseudomallei, a CDC category B biothreat agent. We have previously shown that lipopolysaccharide (LPS) extracted from B. pseudomallei can provide protection against a lethal challenge of B. pseudomallei in a mouse model of melioidosis. Sugar analysis on LPS from B. thailandensis strain E264 confirmed that this polysaccharide has a similar structure to LPS from B. pseudomallei. Mice were immunised with LPS from B. thailandensis or B. pseudomallei and challenged with a lethal dose of B. pseudomallei strain K96243. Similar protection levels were observed when either LPS was used as the immunogen. This data suggests that B. thailandensis LPS has the potential to be used as part of a subunit based vaccine against pathogenic B. pseudomallei.
Article: Melioidosis: the tip of the iceberg?[show abstract] [hide abstract]
ABSTRACT: For nearly 80 years clinical melioidosis has been considered a rare disease. This bacterial infection is caused by Pseudomonas pseudomallei, a saprophyte found in soil and surface water of endemic areas. Consequently, those who have most contact with soil, the rural poor, are likely to be at greatest risk of infection. Since the diversity of clinical manifestations necessitates the isolation and identification of the causative organism for a definitive diagnosis of melioidosis and the population at greatest risk within endemic areas rarely have access to an appropriate level of health care, the disease has probably been underrecognized. Melioidosis is now known to be an important cause of human morbidity and mortality in Thailand, and this may be true throughout Southeast Asia, which is usually regarded as the main endemic area for the disease. In Australia, melioidosis causes a smaller number of human infections, while disease among livestock has important economic and possible public health implications. Sporadic reports of the infection indicate its presence in several other tropical regions: in the Indian subcontinent, Africa, and Central and South America. Clinical melioidosis may be highly prevalent in these areas, but underdiagnosed as a result of a lack of awareness of the clinical and microbiological features of the disease, or simply because of a lack of health care facilities. Furthermore, during the last two decades the importation and transmission of melioidosis within nontropical zones have been documented. The causative organism is not difficult to grow, and modern antibiotics have improved disease prognosis. Further studies are needed to determine the true worldwide distribution and prevalence of melioidosis so that improved therapeutic and preventive measures can be developed and applied.Clinical Microbiology Reviews 02/1991; 4(1):52-60. · 16.13 Impact Factor
Article: The antimicrobial susceptibility of Pseudomonas pseudomallei. Emergence of resistance in vitro and during treatment.[show abstract] [hide abstract]
ABSTRACT: We have measured the in-vitro activity of 27 antimicrobials against 211 clinical and ten reference strains of Pseudomonas pseudomallei. Imipenem was the most active antibiotic tested, followed by piperacillin, doxycycline, amoxycillin/clavulanic acid, cefixime, cefetamet, azlocillin and ceftazidime, all of which had MICs of less than or equal to 2 mg/l for the majority of strains. The measured MICs were dependent on the media and inocula used, to an extent which varied with the antibiotic class under test; MICs of ureidopenicillins were particularly inoculum-dependent. The beta-lactams and ciprofloxacin were bactericidal, whereas the agents conventionally used to treat melioidosis (doxycycline, chloramphenicol, sulphamethoxazole and trimethoprim) had bacteriostatic activity only. Strains highly resistant to chloramphenicol (MIC greater than or equal to 256 mg/l) emerged during treatment in 7.1% of patients. These strains were fully virulent, and frequently showed cross-resistance to tetracyclines, sulphamethoxazole, trimethoprim and ciprofloxacin, with paradoxical increased susceptibility to beta-lactams and aminoglycosides. Similar resistance patterns were seen in mutants generated in vitro and two reference strains. One strain with isolated ceftazidime resistance, reversible by clavulanic acid, emerged during treatment. Several of the new beta-lactam antibiotics are of potential value in the therapy of P. pseudomallei infections. Patients should be carefully monitored for the emergence of antibiotic-resistant strains during treatment of melioidosis.Journal of Antimicrobial Chemotherapy 10/1989; 24(3):295-309. · 5.07 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Melioidosis is an infectious disease that occurs in tropical regions, particularly in Thailand. It is caused by the bacterium Burkholderia pseudomallei and is a serious condition which can be fatal. Beta-lactam antibiotics have dramatically reduced the risk of death, but mortality still remains high. To summarize reliable evidence on the effects of treatment regimens on death and relapse. We searched the Cochrane Infectious Diseases Group trials register (July 2002), the Cochrane Controlled Trials Register (Issue 3, 2002), MEDLINE (1966 to July 2002), EMBASE (1980 to May 2002), BIOSIS (up to July 2002), Health Star (up to July 2002), and reference lists of articles. We also contacted pharmaceutical companies and researchers in the field. Randomized and quasi-randomized controlled trials comparing antibiotic regimens in people with melioidosis. We independently assessed the eligibility of studies and the methodological quality of the trials. Adverse effects information was collected from the trials. Nine trials, all from Thailand, involving a total of 872 participants were included. For intravenous therapy in the acute phase, we identified six trials with a total of 619 participants. Chloramphenicol, doxycycline, and co-trimoxazole (trimethoprim-sulphamethoxazole) combination regimens were associated with a mortality of 50% or more (two studies). Participants randomized to regimens including ceftazidime were more likely to survive (relative risk [RR] 0.46; 95% confidence interval [CI] 0.30 to 0.71). When ceftazidime-containing regimens were compared with beta-lactam or alternative beta-lactamase inhibitor regimens such as co-amoxiclav (amoxycillin-clavulanic acid) and cefoperazone-sulbactam, or with imipenem, mortality rates were similar (RR 1.06; 95% CI 0.81 to 1.39). For oral therapy in the maintenance phase, we found three trials of 253 participants. They compared the conventional regimen (chloramphenicol, doxycycline, and trimethoprim-sulphamethoxazole) with other regimens (amoxycillin-clavulanic acid, ciprofloxacin-azithromycin, and doxycycline alone). There were fewer deaths with the conventional regimen, but no statistically significant differences demonstrated. Regimens for the acute phase of illness should contain ceftazidime or imipenem. It is not yet clear if combinations of treatments in the early phase reduce relapse. For oral therapy after the acute phase of treatment, trials suggest that conventional four drug regimens can be used for treatment.Cochrane database of systematic reviews (Online) 02/2002; · 5.72 Impact Factor
Vaccine 28 (2010) 7551–7555
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/vaccine
Lipopolysaccharide from Burkholderia thailandensis E264 provides protection in a
murine model of melioidosis
Sarah A. Ngugia, Valeria V. Venturab, Omar Qazic, Sarah V. Hardinga, G. Barrie Kittoc,d,
D. Mark Estese, Anne Dellb, Richard W. Titballf, Timothy P. Atkinsa, Katherine A. Brownb,c,d,
Paul G. Hitchenb,g, Joann L. Priora,∗
aDstl Porton Down, Salisbury, Wiltshire SP4 0JQ, UK
bDepartment of Life Sciences, Imperial College London, South Kensington Campus, Exhibition Road, London SW7 2AZ, UK
cInstitute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA
dDepartment of Chemistry and Biochemistry, University of Texas at Austin, Austin, TX 78712, USA
eDepartment of Microbiology and Immunology, Department of Pathology and the Sealy Center for Vaccine Development, University of Texas Medical Branch,
Galveston, TX 77555, USA
fUniversity of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK
gCentre for Integrative Systems Biology, Imperial College London, South Kensington Campus, Exhibition Road, London SW7 2AZ, UK
a r t i c l e i n f o
Received 24 May 2010
Received in revised form 10 August 2010
Accepted 11 August 2010
Available online 15 September 2010
a b s t r a c t
Burkholderia thailandensis is a less virulent close relative of Burkholderia pseudomallei, a CDC category B
biothreat agent. We have previously shown that lipopolysaccharide (LPS) extracted from B. pseudomallei
to LPS from B. pseudomallei. Mice were immunised with LPS from B. thailandensis or B. pseudomallei and
challenged with a lethal dose of B. pseudomallei strain K96243. Similar protection levels were observed
to be used as part of a subunit based vaccine against pathogenic B. pseudomallei.
Crown Copyright © 2010 Published by Elsevier Ltd. All rights reserved.
Burkholderia pseudomallei is the causative agent of melioido-
sis, a severe infectious disease of humans and animals which is
reported most frequently in SE Asia and Northern Australia . The
disease is difficult to treat with antibiotics because of the intrin-
sic antibiotic resistance of the bacterium [2,3], and there is no
licensed vaccine available for use in humans. Because of the abil-
ity of the bacterium to infect via the respiratory tract, it is also
considered to be a potential bioweapon . Studies in Thailand
have shown that the bacterium can be readily isolated from the
soil in melioidosis endemic areas . In addition, the related bac-
terium Burkholderia thailandensis, can often be isolated from soil
in these areas. The clearest distinction between these two species
is the ability of B. thailandensis to assimilate l-arabinose (Ara+),
whereas B. pseudomallei (Ara−) does not have this capability, and
in fact lacks the entire arabinose assimilation operon . Addition-
ally, B. thailandensis is considered to be avirulent in humans, with
∗Corresponding author. Tel.: +44 01980 614739; fax: +44 01980 614307.
E-mail address: JLPRIOR@dstl.gov.uk (J.L. Prior).
a median lethal dose (MLD) in hamsters of approximately 1×106
bacteria (48h post-inoculation), when given intra-peritoneally (IP)
 and approximately 1×109bacteria (IP) in BALB/c mice . In
contrast, B. pseudomallei is highly virulent in Syrian hamsters hav-
ing an MLD of <10 bacteria (IP), and in BALB/c mice (IP) an MLD of
approximately 100 bacteria [6,7].
Lipopolysaccharide (LPS) is a major component of the outer
membrane of Gram-negative bacteria. It is composed of three main
regions, lipid A, the core and multiple O-antigen units covalently
linked to one another. Lipid A anchors the LPS in the outer mem-
brane, the core is a mixture of sugars and sugar derivatives, whilst
the O-antigen is a polysaccharide of repeating oligosaccharide
units that extends from the cell surface, and typically determines
serotype specificity [8,9]. There are three types of B. pseudomallei
LPS. The predominant type (97%) is known as type A or typical,
whilst the less common LPS (2%) is known as type B or atypical.
Both are smooth types of LPS, whereas the remaining 1% lacking
an O-antigen is termed rough LPS. When LPS separated by sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
is stained with silver, one of three different profiles is seen. A lad-
der for typical and atypical LPS, whilst no ladder is observed for the
rough strains . The cellular fatty acid profiles and lipid A struc-
0264-410X/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Ltd. All rights reserved.
S.A. Ngugi et al. / Vaccine 28 (2010) 7551–7555
tures of B. pseudomallei and B. thailandensis have been described
[11,12]. It was established that these species share the same fatty
present in B. pseudomallei [11,12].
provide protection against an otherwise lethal challenge of B. pseu-
domallei in a mouse model of melioidosis. In mice immunised with
LPS from B. pseudomallei strain K96243, 50% survival was seen at
100% mortality at day 11 and a MTTD of 2.6 days in naïve mice .
which makes the purification of LPS a complex and potentially haz-
ardous procedure. It has been reported that B. thailandensis and B.
heteropolymer of disaccharide repeats with the structure, 3-?-d-
glucopyranose-(1→3)-6-deoxy-?-l-talopyranose-(1→), in which
their 6-deoxy-?-l-talopyranosyl residues are variably substituted
with O-acetyl groups at the O-2 or O-4 positions [14–16]. Further-
more, LPS isolated from B. thailandensis and B. pseudomallei show
similar banding patterns on silver-stained SDS-PAGE gels, and in
mice or rabbits [17,18]. The similarity of these LPSs may suggest
that it would be possible to immunise mice with LPS extracted
from B. thailandensis and protect them against a lethal challenge
of B. pseudomallei. The extraction of LPS from the BSL-2 bacterium
B. thailandensis would be less expensive, have reduced safety risks
and be more amenable to scale-up compared to extraction from B.
In this work we extracted the LPS from either B. pseudomallei
K96243 or B. thailandensis E264, and analysed them by silver stain-
protective antigens in a murine melioidosis model of infection, and
determined the antibody isotype of the serum antibody immune
2. Materials and methods
2.1. LPS extraction and gel electrophoresis
LPS was extracted from B. thailandensis strain E264 and B. pseu-
methods [19,20]. In brief, cell pellets of B. pseudomallei K96243
and B. thailandensis E264 were digested for 16h at 4◦C with
15,000Units of lysozyme (Sigma, UK) per mg of bacteria, prior to
digestion with 20?g/ml of DNase I and RNase A (Sigma, UK) for a
further 16h at room temperature. A modified hot phenol method
and visualised by silver staining .
2.2. Chemical derivatisation to alditol acetates for GC–MS
For compositional analysis of the O-antigen by alditol acetate,
the LPS samples were hydrolysed (2M TFA, 121◦C, 2h) and the
monosaccharides reduced (10mg/ml NaBD4room temperature,
2h). After borate removal by repeated evaporation (4×) with 10%
acetic acid in methanol, the sugars were re-acetylated (acetic
anhydride, 100◦C, 1h) and prepared for GC–MS by suspension in
2.3. GC–MS analysis
GC–MS analysis was carried out on a Clarus 500 GC–MS instru-
ment (Perkin-Elmer, USA). Samples were dissolved in hexanes
and injected on a RTX-5MS fused silica column (30mm×0.25mm
Fig. 1. Silver-stained SDS-PAGE of LPS isolated from B. pseudomallei strain K96243
(lane 1) or B. thailandensis E264 (lane 2).
tives, the oven was held at 60◦C for 1min and increased to 190◦C
at 20◦Cmin−1, at which point the temperature was increased to
raised at 25◦Cmin−1and held for a total of 5min.
2.4. LPS protective efficacy
Groups of six female BALB/c mice, aged 6–8 weeks (Charles
River Laboratories, Kent, UK) were immunised with purified LPS
extracted from B. thailandensis strain E264 or B. pseudomallei strain
K96243. On days 0, 14 and 28 mice were dosed intra-peritoneally
(IP) with 10?g of LPS in PBS. Mice were tail-bled 4 weeks after
the final immunisation. Blood for each group was pooled and sera
last immunisation with 55 MLD’s of B. pseudomallei strain K96243
by the IP route, delivered in 0.1ml. All procedures were performed
ers and spleens were harvested from surviving mice for bacterial
2.5. Serum response to LPS
Levels of IgG1, IgG2a, IgG3 and IgM were measured from sera
collected 4 weeks post-final vaccination by ELISA. Microtitre plates
(Immulon 2HB, USA) were coated using anti-mouse fab-specific
S.A. Ngugi et al. / Vaccine 28 (2010) 7551–7555
Fig. 2. GC–MS alditol acetate sugar analysis of hydrolysed and derivatised LPS from (a) B. pseudomallei and (b) B. thailandensis. Non-sugar impurities are labelled X.
antibody (Sigma, UK) at 5?g/ml in PBS and B. pseudomallei K96243
LPS at 5?g/ml, both100?l/well. Plates were washed three times
with 0.05% (v/v) PBS/Tween (Oxoid, UK/Sigma, UK) using an ELx
405 96-well automated plate washer (BioTek Instruments, UK),
and blocked with 200?l/well, 2% (w/v) skimmed milk powder
(Blotto) for 1h at 37◦C. Plates were re-washed as previously
described. A standard curve was prepared and pooled serum sam-
ples diluted 1:100 in Blotto. Diluted naïve sera were included on
each plate as a negative control. Standards and samples were seri-
ally diluted. Plates were incubated for 1h at 37◦C and re-washed.
100?l of HRP-conjugated isotype-specific goat anti-mouse anti-
body, (AbDserotec, UK) was added to each well and incubated at
37◦C for 1h. Plates were washed as previously. 100?l of ABTS
substrate (Sigma, UK) was added to each well; plates were incu-
bated for 15min at 37◦C and read on a Multiskan Ascent plate
reader (Thermo Life sciences, UK) at 414nm. Analysis was car-
ried out using ELISA for Windows version 2.0 (freely available
from http://www.cdc.gov.ncidod/dbmd/bimb/elisa.htm). This uses
a four-parameter logistic fit curve to calculate unknown sample
concentrations of antibody in ?g/ml.
3. Results and discussion
We have shown that LPS extracted from B. thailandensis strain
E264 provides protection against a lethal challenge of B. pseudoma-
llei strain K96243 by the IP route and produces a specific antibody
3.1. LPS profiles
When visualised with silver-staining the LPS extracted from B.
thailandensis shows a characteristic LPS ladder banding pattern. It
S.A. Ngugi et al. / Vaccine 28 (2010) 7551–7555
has a profile comparable to the LPS extracted from B. pseudomallei
strain K96243, displaying very similar spacing between O-antigen
units (Fig. 1).
3.2. Sugar composition of B. thailandensis LPS
The purified LPS samples of B. pseudomallei and B. thailandensis
were analysed for sugar composition as alditol acetate-derivatised
monosaccharides to determine their sugar compositions (Fig. 2).
GC–MS analysis revealed close similarities between the B. pseu-
domallei and B. thailandensis O-antigens, although their talose
methylation patterns are not identical.
Both LPSs were found to contain talose and glucose in their
O-antigens which is consistent with previous reports [14–16]. In
addition, the GC–MS spectra contain mannoheptose and rhamnose
which are likely to be derived from the core of the LPS. Differ-
ences in the LPSs were associated with the talose sugar. Thus B.
pseudomallei LPS possessed both methylated and unmethylated
talose, in contrast to the B. thailandensis sample which had only
unmodified talose. The two talose forms in the B. pseudomallei LPS
chromatograms are consistent with the published literature which
has reported that 33% of the talose residues in B. pseudomallei LPS
are 2-O-methylated [15,16]. Our data suggests that the major dif-
ference between the two O-antigens is the absence of methylated
talose in B. thailandensis.
Additional minor components were observed in the B. pseudo-
mallei alditol acetate chromatogram, which are likely associated
with the LPS core and/or contaminating polymers. For exam-
ple a minor deoxymannoheptose peak was observed in the B.
pseudomallei LPS chromatogram, but not in the B. thailandensis
data. This is probably derived from trace amounts of co-purified
capsular polysaccharide which is known to be an unbranched
homopolymer of O-acetylated deoxymannoheptose. The absence
of the deoxymannoheptose sugar in the B. thailandensis LPS anal-
ysis is consistent with the lack of a capsule in this species . In
addition, some galactose is present in B. pseudomallei LPS but not
B. thailandensis LPS. This is likely to have originated from the LPS
does not contain galactose [14,15].
3.3. Protective efficacy of LPS in a murine model of melioidosis
Despite the difference in the extent of methylation of the talose
monosaccharide between the species, we found, via the experi-
ments described below, that B. thailandensis LPS is able to induce a
protective response against infection with a typical B. pseudomallei
B. pseudomallei was able to induce protection against a subsequent
3 days there were no survivors in the naïve group. At the end of
the experiment (day 35 post-challenge) there was 50% survival of
the mice immunised with B. thailandensis LPS compared with 66%
survival of mice immunised with B. pseudomallei LPS. The mean
time to death (MTTD) for naïve mice was 2.2 days, whereas the
MTTD of mice immunised with B. thailandensis or B. pseudomallei
was 32.8 days or 31.5 days, respectively. There was a statistically
to the control (p<0.001) using a Log rank test. This was repeated
with a challenge of 24 MLD’s of B. pseudomallei strain K96243 (data
not shown). All naïve mice succumbed by day 18, compared to 50%
survival in those mice immunised with B. thailandensis LPS at the
end of the experiment (day 35 post-challenge). The MTTD for naïve
B. thailandensis LPS. The MTTD for naïve mice would be expected
to be higher than observed in the previous experiment due to the
lower challenge dose.
Fig. 3. Survival of BALB/c mice immunised with LPS isolated from B. pseudoma-
llei (O), B. thailandensis (X) or naïve controls (?). Mean time to death in both test
groups was significantly different to the controls (p<0.001) using a Log rank test for
melioidosis endemic areas, and the subsequent development of an
antibody response without overt infection may provide protection
against melioidosis . Immunisation with live B. thailandensis
has reportedly provided 50% protection in guinea pigs when chal-
lenged with 200 LD50B. pseudomallei strain 100 . Moreover,
BALB/c mice immunised IP with 1×108cfuml−1of heat killed B.
thailandensis, and subsequently challenged IP with 40 MLD’s B.
pseudomallei K96243 also showed 50% survival. Additionally, all
spleens were free from signs of infection . In this study liv-
ers and spleens were harvested from all surviving mice immunised
with the LPSs to determine bacterial load. Colonies showing the
ered from three of the spleens, and all four of the livers from the
also recovered from two of the spleens, and all of the livers of the
three surviving animals immunised with LPS from B. thailandensis,
thus mice vaccinated with LPS do not clear the bacteria and may
ultimately succumb to infection.
3.4. Serum antibody responses to LPS immunisation
LPS is a T-independent antigen, and as expected IgM and IgG3
responses predominated in the serological profile of the host
response to vaccination. Mice immunised with LPS from B. thai-
landensis or B. pseudomallei had measurable antibody response in
IgG3 and IgM for both LPS’.
In conclusion, consistent with previous studies using B. pseudo-
mallei LPS we have demonstrated that B. thailandensis LPS is both
structurally similar, and displays a comparable level of partial pro-
tection against melioidosis in a mouse model of infection . This
offers the possibility of using B. thailandensis LPS or LPS moieties
in vaccines targeted to B. pseudomallei. For example to improve
the efficacy of a vaccine, the LPS could be conjugated to a carrier
protein to generate a T-cell dependent response . Previously
B. pseudomallei LPS has been conjugated to tetanus toxoid or flag-
ellin to produce a glycoconjugate. The passive transfer of antisera
against these glycoconjugates conferred protection to diabetic rats
against B. pseudomallei strain 316c [26,27]. Given the results pre-
sented in this manuscript, it is likely that similar results could be
obtained using B. thailandensis LPS. Furthermore, the reduced costs
and hazards associated with the production of B. thailandensis LPS
suggest that this molecule can be exploited in a wider range of
studies aimed at developing a vaccine against melioidosis infec-
A.D. and P.G.H. are supported by the Biotechnology and
Biological Sciences Research Council (grants BBF0083091 and
S.A. Ngugi et al. / Vaccine 28 (2010) 7551–7555
BBC5196701 CISBIC). V.V.V. is supported by a student bursary
from Dstl. This work was also supported by NIH/NIAID grants
1U01AI078008-1 and U54AI057156 (KAB, DME, AGT).
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