In vitro susceptibility of Burkholderia pseudomallei to antimicrobial peptides.

Abstract

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many antibiotics, resulting in high mortality rates of 19% in Australia and even 50% in Thailand. Antimicrobial peptides (AMPs) possess potent broad-spectrum bactericidal activities and are regarded as promising therapeutic alternatives in the fight against resistant microorganisms. Moreover, these peptides may also affect inflammation, immune activation and wound healing. In this study, the in vitro activities of 10 AMPs, including histatin 5 and histatin variants, human cathelicidin peptide LL-37 and lactoferrin peptides, against 24 isolates of B. pseudomallei were investigated. The results showed that the antibacterial activities of the individual peptides depended on peptide dose and bacterial isolate. Among the 10 peptides tested, LL-37 exhibited the most effective killing activity. The smooth type A lipopolysaccharide (LPS) phenotype B. pseudomallei appeared to be more susceptible than those expressing the smooth type B LPS and the rough type LPS. Four isolates of B. pseudomallei shown to be resistant to ceftazidime and trimethoprim/sulfamethoxazole were also highly susceptible to LL-37. These data indicate that LL-37 possesses antimicrobial activity against all isolates independent of the LPS phenotype and is therefore a promising peptide to combat B. pseudomallei infections.

Full text

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International Journal of Antimicrobial Agents 34 (2009) 309–314
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents
journal homepage: http://www.elsevier.com/locate/ijantimicag
In vitro susceptibility of Burkholderia pseudomallei to antimicrobial peptides
Sakawrat Kanthawonga,b, Kamran Nazmic, Surasakdi Wongratanacheewina,b, Jan G.M. Bolscherc,
Vanaporn Wuthiekanund, Suwimol Taweechaisupapongb,e,∗
aDepartment of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
bMelioidosis Research Center, Khon Kaen University, Khon Kaen 40002, Thailand
cDepartment of Oral Biochemistry, Academic Centre for Dentistry, Amsterdam, University of Amsterdam and VU University Amsterdam, 1081 BT Amsterdam, The Netherlands
dFaculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand
eDepartment of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand
a r t i c l ei n f o
Article history:
Received 12 February 2009
Accepted 26 May 2009
Keywords:
Burkholderia pseudomallei
Antimicrobial peptides
Histatins
LL-37
Lactoferrin
a b s t r a c t
Burkholderiapseudomallei,thecausativeagentofmelioidosis,isintrinsicallyresistanttomanyantibiotics,
resulting in high mortality rates of 19% in Australia and even 50% in Thailand. Antimicrobial peptides
(AMPs) possess potent broad-spectrum bactericidal activities and are regarded as promising therapeu-
tic alternatives in the fight against resistant microorganisms. Moreover, these peptides may also affect
inflammation, immune activation and wound healing. In this study, the in vitro activities of 10 AMPs,
including histatin 5 and histatin variants, human cathelicidin peptide LL-37 and lactoferrin peptides,
against 24 isolates of B. pseudomallei were investigated. The results showed that the antibacterial activ-
ities of the individual peptides depended on peptide dose and bacterial isolate. Among the 10 peptides
tested, LL-37 exhibited the most effective killing activity. The smooth type A lipopolysaccharide (LPS)
phenotype B. pseudomallei appeared to be more susceptible than those expressing the smooth type B LPS
andtheroughtypeLPS.FourisolatesofB.pseudomalleishowntoberesistanttoceftazidimeandtrimetho-
prim/sulfamethoxazole were also highly susceptible to LL-37. These data indicate that LL-37 possesses
antimicrobial activity against all isolates independent of the LPS phenotype and is therefore a promising
peptide to combat B. pseudomallei infections.
© 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction
Burkholderia pseudomallei is the causative agent of melioido-
sis, a severe disease endemic in areas of Southeast Asia, Northern
Australia, the Indian subcontinent, Iran, and Central and South
America [1,2]. It is an environmental saprophyte that can be iso-
lated from soil and water in these endemic areas and is known
as a potential biological warfare agent. Infection is thought to be
acquiredeitherthroughawoundintheskinorthroughinhalationof
aerosolised B. pseudomallei. Melioidosis may arise many years after
exposure, commonly in association with compromised immunity.
Overall mortality is 50% in Northeast Thailand (35% in children) [1]
and 19% in Australia [2]. Recurrent infection is the most important
complication in survivors despite 20 weeks of antimicrobial treat-
ment and occurs in 10% of Thai patients who survive the primary
episode [3].
Burkholderia pseudomallei is intrinsically resistant to many
antibiotics, including penicillin, first- and second-generation
∗Corresponding author. Present address: Department of Oral Diagnosis, Faculty
ofDentistry,KhonKaenUniversity,KhonKaen40002,Thailand.Fax:+6643202862.
E-mail address: suvi taw@kku.ac.th (S. Taweechaisupapong).
cephalosporins, macrolides, rifamycins, colistin and aminogly-
cosides, but is usually susceptible to amoxicillin/clavulanic
acid (AMC), chloramphenicol, doxycycline, trimethoprim/sulfa-
methoxazole (SXT), ureidopenicillins, ceftazidime and carbapen-
ems [1,2]. Ceftazidime, the carbapenem antibiotics or AMC are
used for the initial parenteral phase of therapy, followed by a pro-
longed course of oral antimicrobial therapy with either SXT with
or without doxycycline or AMC [4]. However, ceftazidime- and/or
AMC-resistant B. pseudomallei have emerged, ultimately leading to
treatment failure [5–7]. The carbapenems have been reported to
have good bactericidal activities against B. pseudomallei and have
been used effectively to treat patients with septicaemic melioido-
sis [8,9]. However, increased use of carbapenems may again give
rise to resistance as has been seen with ceftazidime and AMC.
Owing to the difficulty in eradication of the organism following
infection,prolongedantibiotictherapyisneededandahighrelapse
rate is observed if therapy is not completed [10]. Furthermore,
recurrence of infection is common despite adequate antimicrobial
therapy [2]. It has been shown that B. pseudomallei isolated from
relapse cases and persistent infections were resistant to antibiotics
[6,11]. With the increasing development of antibiotic resistance,
it is necessary to search for novel anti-infective agents against B.
pseudomallei.
0924-8579/$ – see front matter © 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
doi:10.1016/j.ijantimicag.2009.05.012

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Table 1
Sequences and characteristics of the peptides investigated.
PeptideSequence Mol. wt. Chargea
Histatin 5
dh5
dhvar2
dhvar3
dhvar4
dhvar5
bLFcin17-30
bLFampin265-284
bLFampin268-284
LL-37
DSHAKRHHGYKRKFHEKHHSHRGY
KRKFHEKHHSHRGY
KRLFKELLFSLRKY
KRLFKKLKFSLRKY
KRLFKKLLFSLRKY
LLLFLLKKRKKRKY
FKCRRWQWRMKKLG
DLIWKLLSKAQEKFGKNKSR
WKLLSKAQEKFGKNKSR
LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES
3036
1847
1841
1855
1840
1847
1922
2389
2047
4494
5+
4+
4+
7+
6+
7+
6+
4+
5+
6+
aNet positive charge at neutral pH.
Antimicrobial peptides (AMPs) have received special atten-
tion as a possible alternative means to combat antibiotic-resistant
bacterialstrains.Theyareattractivecandidatesforclinicaldevelop-
ment because of their selectivity, their speed of action and because
bacteria may not easily develop resistance against them. In the last
few decades, an increasing number of AMPs have been isolated
and characterised from virtually all classes of organisms where
they play an important role in the innate defence against micro-
bial and viral infections. In addition to their antimicrobial role,
these peptides may also serve as important effector molecules in
inflammation, immune activation and wound healing [12–14]. The
AMPsdiscoveredsofarhavebeendividedintoseveralgroupsbased
on their length, secondary and tertiary structure, and presence or
absence of disulfide bridges [15]. Most of them share common fea-
turessuchassmallsize,cationicchargeandanamphipathicnature.
Several cationic peptides bind to the negatively charged residues of
lipopolysaccharide (LPS) of the outer membrane by electrostatic
interactions involving the negatively charged phosphoryl groups
andbyhydrophobicinteractionsinvolvingtheacylchainsoflipidA,
thusdestabilisingthemicrobialmembraneandleadingtocelldeath
for Gram-negative organisms [16,17]. However, different modes of
action are proposed for several peptides, including inhibition of
the synthesis of specific membrane proteins [18] or stress proteins
[19], arrest of DNA synthesis [20], interaction with DNA [21] and
production of hydrogen peroxide [22].
TheAMPsLL-37,histatin5andhistatinvariants,andthelactofer-
rin peptides have been proven to be active against fungi as well as
Gram-positive and -negative bacteria [23–27]. To identify the most
effective peptides against B. pseudomallei, we compared the in vitro
antimicrobial activities of histatin 5 and its variants, LL-37 and the
lactoferrin peptides against 24 isolates of B. pseudomallei. Possible
association between the LPS type and susceptibility of B. pseudo-
mallei to these AMPs was also investigated. The results reported in
this study indicate that, of the tested peptides, LL-37 exhibited the
most effective killing activity and possessed antimicrobial activity
against all isolates independent of the LPS phenotype. Therefore, it
is a promising peptide to combat B. pseudomallei infections.
2. Materials and methods
2.1. Synthetic peptides
Histatin 5 and its variants (dh5, dhvar2, dhvar3, dhvar4 and
dhvar5), the lactoferrin peptides LFcin17-30, LFampin268-284
and LFampin265-284, corresponding to the amino acids 17–30,
268–284 and 265–284 of bovine lactoferrin (bLF), respectively, and
the human cathelicidin peptide LL-37 were synthesised by fluoren-
9-ylmethoxycarbonyl (Fmoc) chemistry with a MilliGen 9050
peptidesynthesizer(MilliGen-Biosearch,Bedford,MA)asdescribed
previously [23,26,27]. All peptides were ≥95% pure as deter-
mined by reverse-phase high-performance liquid chromatography
(RP-HPLC) (JASCO Corp., Tokyo, Japan). The authenticity of the pep-
tides was confirmed by ion-trap mass spectrometry with an LCQ
DecaXP(ThermoFinnigan,SanJosé,CA).Aminoacidsequencesand
characteristics of the 10 peptides investigated are shown in Table 1.
2.2. Bacteria and growth conditions
Twenty-four isolates of B. pseudomallei were used in this study
(Table 2), 13 of which were isolated from patients admitted to Sri-
nagarindHospital(FacultyofMedicine,KhonKaenUniversity,Khon
Kaen, Thailand) and 11 were isolated from soil collected from the
northeastern endemic region of Thailand. Individual isolates were
identified by biochemical tests, antibiotic susceptibility profile and
immunoreactivity with polyclonal and monoclonal antibodies as
described elsewhere [28–30]. The B. pseudomallei isolates were
divided into three groups based on their LPS phenotype (smooth
type A, smooth type B and rough type) as described previously[31].
The media used in this study were brain–heart infusion (BHI)
broth (Difco, Becton Dickinson Microbiology Systems, Franklin
Lakes,NJ)andnutrientagar.Burkholderiapseudomalleiisolateswere
cultured aerobically in BHI broth at 37◦C overnight and, to yield
a mid logarithmic growth phase, were subcultured at 37◦C in a
200rpm shaker-incubator for 1.5h.
2.3. Antibacterial assay
To determine the effective concentration of peptides against B.
pseudomallei,variousconcentrationsofhistatin5andLFampin268-
284 were tested against B. pseudomallei isolate A2, which is a
virulent strain with a 50% lethal dose of 19 in BALB/c mice [32].
Bacterial cells were washed three times and re-suspended to ca.
1×107colony-forming units (CFU)/mL in 1mM potassium phos-
phate buffer (PPB) (pH 7.0). The bacterial suspension was then
added to an equal volume of the AMP to reach a final concentration
Table 2
Source and lipopolysaccharide (LPS) phenotype of clinical and environmental
Burkholderia pseudomallei isolates in this study.
LPSsmoothtypeA LPS smooth type A LPSsmoothtypeBLPS rough type
Isolate Source IsolateSource IsolateSource IsolateSource
267
279
354
377
409
429
466
591
705
745
844
Soil, NE
Soil, NE
Soil, NE
Soil, NE
Soil, NE
Soil, NE
Soil, NE
Soil, NE
Soil, NE
Soil, NE
Soil, NE
A2
316c
FL202
979b
2-173
365a
Blood
Blood
Fluid
Sputum
Urine
Urine
A1
A20
G12
U2704
Blood
Blood
Pus
Urine
316a
U882b
G207
Blood
Pus
Sputum
NE, northeast region of Thailand.

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311
of 10–100?M. Bacterial suspension in PPB without peptide served
as a control. Following incubation at 37◦C for 60min, the incuba-
tion mixture was serially diluted in sterile normal saline and plated
intriplicateonnutrientagar.Colonieswerecountedafter20hincu-
bation at 37◦C. The percentage killing or inhibiting effects of each
AMP were calculated using the formula [1−(CFU sample/CFU con-
trol)]×100%.
To compare the in vitro activities of the 10 peptides against
the 24 isolates of B. pseudomallei, a final concentration of 100?M
of peptide was chosen based on the maximum killing activity in
the previous initial tests. The antibacterial assay was performed as
described above.
2.4. Killing kinetic assay of the most effective antimicrobial
peptide
Killing kinetics was determined using a culture of B. pseudo-
mallei
isolateA2re-suspended
1×105CFU/mL. Peptide was added to the bacterial suspension
to a final concentration of 200?M, which was the minimum
bactericidal concentration (MBC), and incubated in a 200rpm
shaker-incubatorat37◦C.Attheindicatedtimes(0,1,2,4,6,8,10,12
and 24h), samples were taken, serially diluted, plated in triplicate
on nutrient agar and incubated for 24h to allow colony counting.
A bactericidal effect was defined as a ≥3log10reduction in CFU/mL
compared with the initial inoculum.
in1mM PPBto give ca.
3. Results
3.1. Antibacterial effect of the peptides
To obtain initial information on the susceptibility of B. pseudo-
mallei to AMPs, a test was performed with different concentrations
of histatin 5 and LFampin268-284. Both peptides effected a clear
dose-dependent killing activity against B. pseudomallei isolate A2
(Fig. 1). They exhibited 50% killing activities at a peptide concen-
tration of 75?M and 50?M, respectively. Based on this result, the
antibacterial activities of 10 peptides were determined at a concen-
tration of 100?M against 24 isolates of B. pseudomallei of different
originanddistinctLPStypes(smoothtypesAandBandroughtype).
The antimicrobial activities of the 10 peptides were largely isolate
dependent. Of note, only LL-37 caused killing towards virtually all
isolates (Fig. 2); 80–100% killing was observed for all soil isolates
Fig. 1. Dose–response of the bactericidal activity of histatin 5 and LFampin268-284
against Burkholderia pseudomallei isolate A2. A bacterial suspension of ca. 1×107
colony-forming units (CFU)/mL was incubated with each antimicrobial peptide at
final concentrations of 10–100?M at 37◦C for 1h. Subsequently, the incubation
mixture was serially diluted, plated in triplicate on nutrient agar and incubated at
37◦C. After 18h, colonies were counted and the percentage killing was calculated
using the formula [1−(CFU sample/CFU control)]×100%.
and75%toalmost100%killingwasobservedfortheclinicalisolates
with the exception of the 2 of 13 isolates that exhibited killing of
ca. 50% and 60% (isolates 2-173 and A20, respectively).
Isolate-dependent killing of the other peptides can best be illus-
tratedonthebasisofselectedexamplesforthesoilisolates(Fig.2A).
(i)Theoverallmostsensitivesoilisolate377turnedouttobemainly
susceptible to the dhvar series of peptides but not or much less
susceptible to the parental histatin 5 or its active domain dh5,
whilst soil isolate 466 appeared to be susceptible to dhvar4 and
much less to the other variants. (ii) With respect to the lactoferrin
peptides, isolate 377 was sensitive to LFcin17-30 and LFampin265-
284 but not to LFampin268-284, which is only three amino acids
smaller. The opposite was found for soil isolate 354, which was
more susceptible to LFampin268-284 than to LFampin265-284 or
LFcin17-30. However, another soil isolate, 745, appeared equally
sensitive to both LFampins and absolutely insensitive to LFcin17-
30. Similar examples can be given for the clinical isolates (Fig. 2B).
(i) The most sensitive clinical isolates were A2 and FL202. Isolate
FL202 expressed high sensitivity to histatin 5 and dh5 whilst hav-
ingalowersensitivitytodhvar2andagainincreasingsensitivitiesto
dhvar3, dhvar4 and dhvar5. Isolate 979b appeared to be most sus-
ceptibletodhvar3anddhvar4,withlesssensitivitytotheothers.(ii)
Isolate FL202 also appeared to be very sensitive to the lactoferrin
peptides LFcin17-30 and LFampin265-284 but less to LFampin268-
284. Similar to the soil bacteria, another isolate (316c) also showed
theoppositeeffect,withhighestsensitivitytoLFampin268-284and
much less to the other two lactoferrin peptides.
If the tested B. pseudomallei isolates are compared with respect
to their LPS types, e.g. smooth types A and B and rough type, it
is clear that B. pseudomallei with LPS of the smooth type A was
more susceptible to all the peptides tested than those with smooth
type B or rough type LPS (Fig. 3). The average percentage killing
activities of LL-37 against the smooth types A and B and the rough
type B. pseudomallei were ca. 94%, 77% and 83%, respectively. For
histatin 5 and its variants, the average percentage killing activities
againstthesmoothtypesAandBandtheroughtypeB.pseudomallei
were 41–55%, 24–42% and 16–36%, respectively, whilst the average
percentage killing activities of lactoferrin peptides were 37–45%,
21–31% and 16–33%, respectively.
Four of the tested B. pseudomallei isolates (316c, 365a, 979b
and FL202) of smooth type A LPS tested in our laboratory and by
others [33,34] were resistant to the classic antibiotics ceftazidime
and SXT, at present the treatment of choice for severe melioido-
sis. The minimum inhibitory concentrations of ceftazidime and
SXT towards these isolates (Table 3) are clearly beyond the break-
points for resistance to ceftazidime and SXT in B. pseudomallei,
adapted from data compiled by the National Committee for Clinical
Laboratory Standards for similar organisms to be used for suscepti-
bility testing [35]. To emphasise the antimicrobial activity of LL-37
against the antibiotic-resistant B. pseudomallei, the killing activities
of all the peptides are given in Fig. 4. It is interesting and promis-
ing that although resistant to ceftazidime and SXT, these isolates
were highly susceptible to LL-37, whereas the other peptides again
showed isolate selectivity to these four isolates.
Table 3
Minimum
prim/sulfamethoxazole (SXT) towards Burkholderia pseudomallei isolates.
inhibitoryconcentrations (MICs)of ceftazidimeandtrimetho-
IsolateMIC (?g/mL)
CeftazidimeSXT (1:19)
316c
365a
979b
FL202
64
32
256
2/38
0.5/9.5
1/19
4/761

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Fig. 2. Bactericidal activities of 10 antimicrobial peptides (AMPs) against 24 isolates of Burkholderia pseudomallei. A bacterial suspension of ca. 1×107colony-forming units
(CFU)/mL of 11 isolates from environmental samples (A) and 13 isolates from clinical samples (B) were incubated with 100?M AMP and processed as described in Section
2.3. Percentage killing was calculated using the formula [1−(CFU sample/CFU control)]×100% (mean value of triplicate incubations).
3.2. Killing kinetics of clinical Burkholderia pseudomallei isolate
A2 by LL-37
Using B. pseudomallei isolate A2, the killing kinetics of the most
effective antimicrobial peptide, LL-37, was further investigated at
200?M, which was found to be the MBC (data not shown). The
bactericidal endpoint was reached after 6h of incubation (Fig. 5).
There was a rapid decrease in the number of CFU/mL in the first 2h,
with slower killing after that point. Control incubations without
peptide showed an increase in CFU/mL to >106within 2h and a
further increase to 107CFU/mL in the next 24h. Other peptides
showed initial reduction in CFU/mL, however they never reached a
bactericidal endpoint and B. pseudomallei grew to the control level
after 4–8h (data not shown).
4. Discussion
The results from this study show that although individual AMPs
demonstrated selective antibacterial activity towards distinct iso-
lates,thehumancathelicidinpeptideLL-37possessedstrongkilling
activity towards all B. pseudomallei isolates, as also observed in pre-
vious reports for LL-37 against other bacteria [36]. Although the
exact mechanism by which the peptides kill bacteria is not clearly
understood, it has been shown that LL-37 performs its bactericidal
action by electrostatic binding of its cationic molecules to the outer
surface of the bacterial cell. Insertion of the peptide into the cell
membraneresultsinleakageofthecellcytoplasmintotheextracel-
lular space causing death of the bacterial cell [37]. The reason why
LL-37, in contrast to the other peptides, is active against all tested
B. pseudomallei isolates may be found in the fact that this peptide
is much longer, intrinsically having a variety of domains differing
inchargedistributionandamphipathicconformation.Eachdomain
mayhaveitsownoptimalactivityagainstindividualisolates,whilst
the entire peptide is active against virtually all isolates (Fig. 2).
Indeed,partiallyoverlappingtruncatedpeptidesfromLL-37exhibit
different levels of antimicrobial activity against different bacterial
species[38],includingBurkholderiathailandensis(S.Kanthawonget
al., unpublished data).
For the lactoferrin peptides, the mechanism of antibacterial
action has been only partially clarified. The peptide binds to LPS
in Gram-negative bacteria and to teichoic acid in Gram-positive
Fig. 3. Bactericidal activities of 10 antimicrobial peptides against different
lipopolysaccharide (LPS) phenotypes of Burkholderia pseudomallei. Results repre-
sent the mean bactericidal activities of 17 isolates of B. pseudomallei expressing LPS
of smooth type A, 4 isolates with smooth type B and 3 isolates with rough type LPS.

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313
Fig. 4. Antibacterial activity of the 10 antimicrobial peptides (AMPs) against ceftazidime- and SXT-resistant isolates Burkholderia pseudomallei. A bacterial suspension of ca.
1×107colony-forming units (CFU)/mL was incubated with 100?M AMP and processed as described Section 2.3. Percentage killing was calculated using the formula [1−(CFU
sample/CFU control)]×100% (mean value of triplicate incubations).
Fig. 5. Killing kinetics of LL-37 against Burkholderia pseudomallei isolate A2. A bac-
terial suspension of ca. 1×105colony-forming units (CFU)/mL was incubated with
200?Mofpeptideincubatedina200rpmshaker-incubatorat37◦C.Attheindicated
times (0, 1, 2, 4, 6, 8, 10, 12 and 24h), samples were taken, serially diluted, plated
in triplicate on nutrient agar and incubated at 37◦C for 24h. Colonies were counted
and a bactericidal effect was defined as a ≥ 3log10reduction in CFU/mL compared
with the initial inoculum.
bacteria [39]. From there it is assumed that it is brought to the
cytoplasmic membrane where it exerts its effect by disintegrat-
ing the cytoplasmic membrane. This action can be explained by
the conformational flexibility of the lactoferricin portion in the
lactoferrin molecule that allows it to form an amphipathic struc-
ture in solution [40]. In this study, we found that the antibacterial
activities of bLFampin265-284 and bLFampin268-284 were differ-
ent from that of bLFcin17-30. Our results are in accordance with
several previous studies [26,41]. This probably reflects the fact that
thesepeptides,althoughsharingamphipathicandcationicfeatures,
are strikingly different with respect to amino acid composition and
length as well as structure. In addition, besides peptide properties,
other bacteria-associated features may also play an important role
in the peptide-mediated killing.
For the killing kinetic assay of LL-37 against B. pseudomallei iso-
late A2, there was a rapid decrease in the number of CFU/mL in the
first 2h (a reduction of >95% of the vital bacteria). The remaining
<5% bacteria were killed at a slower rate, possibly owing to inter-
ference of the huge amount of bacterial debris with the peptide
binding to vital bacteria or to scavenging of the peptide by this
debris.
The possible association between LPS type and susceptibil-
ity of B. pseudomallei to the AMPs was also investigated, as LPS
is the major structural component of the outer membrane of
Gram-negative bacteria and shields them from a variety of host
defencefactors,includingAMPs.TheresultsrevealedthatB.pseudo-
mallei smooth type A LPS appeared to be the most susceptible to all
peptides tested compared with the smooth type B and the rough
type B. pseudomallei. We recently reported the LPS heterogeneity
among B. pseudomallei and that the two less abundant LPS pat-
terns (smooth type B and rough type) were found more in isolates
obtained from patients with relapsed melioidosis than from those
with primary infection [31]. Since the modification of LPS struc-
ture has been reported to contribute to AMP resistance in several
pathogens,i.e.PseudomonasaeruginosaandSalmonellaspp.[42,43],
the higher resistance of the smooth type B and rough type B. pseu-
domallei to cationic peptides observed in this study may be related
to modification of the LPS structure and the ability of these two
LPS-containing B. pseudomallei to evade host immunity and cause
relapse melioidosis. In contrast to the other peptides, LL-37 was
capable of killing the smooth type B and rough type B. pseudomallei
almost as well as the smooth type A, strongly affirming the potency
of this peptide.
Since 1989, ceftazidime became the treatment of choice for
severe melioidosis because it was associated with 50% lower
overall mortality than conventional treatment [44]. The anti-
microbial combination SXT is commonly used in combination
with ceftazidime or meropenem, or on its own as maintenance
monotherapy because of its low cost, good efficacy and lower
relapse rate compared with AMC [3]. However, the emergence of
ceftazidime-and/orSXT-resistantclinicalisolatesandthewidedis-
tribution of B. pseudomallei in Southeast Asia increase the risk of
malicious use of resistant strains. In the present study, it is very
interesting that four B. pseudomallei isolates (316c, 365a, 979b and
FL202) that were resistant to ceftazidime and SXT were highly sus-
ceptible to LL-37.
In addition to the potential for direct bactericidal activity, LL-
37 may have diverse and complementary abilities to modulate the
innate immune response to B. pseudomallei. LL-37 has been indi-
cated to stimulate angiogenesis [45], to provoke cell migration
[45] and to accelerate wound closure of the airway epithelium
[46]. It is also able to neutralise LPS [37,47] and in doing so pro-
tects against endotoxic shock [48,49]. Furthermore, LL-37 provokes
cytokinerelease,e.g.interleukin(IL)-8andIL-18secretion[14].This
latterrolehasbeenshowntoplayanessentialpartintheprotective
immune response to the severe form of melioidosis [50]. Because
IL-18 is involved both in Th1 and Th2 functions, the ability of LL-37
toinducethesecretionofthispro-inflammatorymediatorindicates
the role of this peptide in both innate and adaptive immunity.
This in vitro study provides evidence that LL-37 has strong
killing activity against B. pseudomallei, however the mechanism
is still unknown. Thus, further investigations are needed to
study the mechanisms of action and, in relation to its suggested

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immunological effect, LL-37 may become an important future con-
sideration for the treatment of melioidosis.
Funding: This work was funded by the Commission on Higher
Education granting under CHE-RG project, Thailand.
Competing interests: None declared.
Ethical approval: Not required.
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