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Antimicrobial and antibiofilm activity of LL-37 and its truncated variants against Burkholderia pseudomallei
International Journal of Antimicrobial Agents 39 (2012) 39–44
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Antimicrobial and antibiofilm activity of LL-37 and its truncated variants against Burkholderia pseudomallei Sakawrat Kanthawong a,b , Jan G.M. Bolscher c , Enno C.I. Veerman c , Jan van Marle d , Hans J.J. de Soet e , Kamran Nazmi c , Surasakdi Wongratanacheewin a,b , Suwimol Taweechaisupapong b,f,∗ a
Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Melioidosis Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand c Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, 1081 BT Amsterdam, The Netherlands d Department of Electron Microscopy, Academic Medical Center, University of Amsterdam, The Netherlands e Department of Oral Microbiology, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, 1081 BT Amsterdam, The Netherlands f Biofilm Research Group, Department of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand b
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Article history: Received 24 June 2011 Accepted 9 September 2011 Keywords: Burkholderia pseudomallei Antimicrobial peptide LL-37 Truncated variants Biofilm
a b s t r a c t The Gram-negative bacterium Burkholderia pseudomallei is the aetiological agent of melioidosis, which is an endemic disease in tropical areas of Southeast Asia and Northern Australia. Burkholderia pseudomallei has intrinsic resistance to a number of commonly used antibiotics and has also been reported to develop a biofilm. Resistance to killing by antimicrobial agents is one of the hallmarks of bacteria grown in biofilm. The aim of this study was to determine the antimicrobial activity and mechanisms of action of LL-37 and its truncated variants against B. pseudomallei both in planktonic and biofilm form, as LL-37 is an antimicrobial peptide that possessed strong killing activity against several pathogens. Antimicrobial assays revealed that LL-31, a truncated variant of LL-37 lacking the six C-terminus residues, exhibited the strongest killing effect. Time–kill experiments showed that 20 M LL-31 can reach the bactericidal endpoint within 2 h. Freeze-fracture electron microscopy of bacterial cells demonstrated that these peptides disrupt the membrane and cause leakage of intracellular molecules leading to cell death. Moreover, LL-31 also possessed stronger bactericidal activity than ceftazidime against B. pseudomallei grown in biofilm. Thus, LL-31 should be considered as a potent antimicrobial agent against B. pseudomallei both in planktonic and biofilm form. © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Burkholderia pseudomallei is the causative agent of melioidosis, a life-threatening bacterial infectious disease of man and animals in Southeast Asia, Northern Australia, the Indian subcontinent, Iran, and Central and South America [1,2]. Clinical manifestations range from seropositive subclinical infection to acute fatal septicaemia or chronic disease [2]. Infection is thought to be acquired either through a wound in the skin or through inhalation of aerosolised B. pseudomallei. Successful treatment of melioidosis patients is difficult because the pathogen is inherently resistant to a variety of antibiotics, including -lactams, aminoglycosides, macrolides and polymyxins [3,4]. Ceftazidime (CAZ), carbapenems such as imipenem and meropenem, and, to a lesser degree,
∗ Corresponding author. Present address: Biofilm Research Group, Department of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand. Fax: +66 43 202 862. E-mail address: suvi [email protected] (S. Taweechaisupapong).
amoxicillin/clavulanic acid remain the backbone of current initial or intensive-phase melioidosis treatment. Resistance to CAZ and imipenem is rare. The current standard treatment with agents to which B. pseudomallei is susceptible requires 2–4 weeks of parenteral therapy, e.g. with CAZ as initial intensive therapy, followed by 3–6 months of oral eradication therapy, e.g. with trimethoprim/sulfamethoxazole, doxycycline, chloramphenicol or a combination therapy. Although CAZ is the drug of choice that is the most effective for treatment of severe melioidosis, the mortality rate in treated patients is >40% [2]. In addition, B. pseudomallei was reported to form biofilm and microcolonies [5]. Burkholderia pseudomallei growing in biofilm is resistant to several conventional antibiotics [6]. This may be proposed as the cause of relapse cases in melioidosis patients. With the increasing development of antibiotic resistance, it is worth searching for novel anti-infective agents against B. pseudomallei. LL-37, the only human member of the cathelicidin family of antimicrobial peptides (AMPs), has killing activity against a broad range of Gram-positive and Gram-negative bacteria in vitro [7–9]. The inhibitory effects of LL-37 on biofilm formation as well as
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the initial attachment of Staphylococcus epidermidis and Pseudomonas aeruginosa have been reported [10,11]. Recently, we found that LL-37 has strong killing activity against B. pseudomallei [12]. Further investigation of a library of LL-37 fragments with overlapping sequences using Burkholderia thailandensis E264 as a model demonstrated that LL-31, a truncated variant of LL-37 missing the six residues at the C-terminus, displayed the strongest killing effect against B. thailandensis E264 by disrupting the bacterial membrane and subsequent leakage of intracellular components leading to death of the bacterium [13]. Although B. thailandensis is closely related to B. pseudomallei [14], susceptibility to antimicrobial agents differs [15]. Therefore, in the present study, a library of LL-37 fragments was further investigated with B. pseudomallei both in planktonic and biofilm form in comparison with the conventional antibiotic CAZ. 2. Materials and methods 2.1. Peptide synthesis, purification and labelling The human cathelicidin peptide LL-37 and its truncated variants [LL-31, LL-25, LL-19, LL-13, RK-31 (LL 7-37), IG-25 (LL 13-37), RI-19 (LL 19-37), KD-13 (LL 25-37), RK-25 (LL 7-31), IG-19 (LL 13-31), RK-19 (LL 9-25) and IG-13 (LL 13-25)] were synthesised by fluoren-9-ylmethoxycarbonyl (Fmoc) chemistry using a MilliGen 9050 Peptide Synthesizer (MilliGen/BioSearch, Bedford, MA) as described previously [16]. For the fluorescein isothiocyanate (FITC) labelling, peptides were extended with the linker FmocL-␥-aminobutyric acid and, after detachment of the Fmoc group, were labelled overnight at 20 ◦ C with 30-fold excess of FITC in N,Ndiisopropylethylamine/dimethylformamide (DIPEA/DMF) before removal of the side chain protecting groups and simultaneous detachment from the resin support. All peptides were of ≥95% purity as determined by reverse-phase high-performance liquid chromatography (RP-HPLC) (JASCO, Tokyo, Japan). The sequences of all peptides investigated are shown in Fig. 1. 2.2. Bacterial strains and growth conditions Burkholderia pseudomallei isolate 1026b as well as H777 (wildtype) and a biofilm-defective mutant (M10) [17] were used in this study. Burkholderia pseudomallei isolate 1026b was kindly provided by Dr W.J. Wiersinga [Center for Infection and Immunity (CINIMA), Center for Experimental and Molecular Medicine, University of Amsterdam, Academic Medical Centre, Amsterdam, The Netherlands] with permission of Prof. Dr D.E. Woods (Department of Microbiology and Infectious Diseases, University of Calgary Health Science Center, Calgary, Alberta, Canada). Burkholderia pseudomallei isolate H777 was isolated from a patient admitted to Srinagarind Hospital (Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand). Media used in this study were brain–heart infusion (BHI) broth (Difco, Becton Dickinson Microbiology Systems, Franklin Lakes, NJ), Luria–Bertani (LB) agar (Difco), Muller–Hinton broth (CriterionTM ; Hardy Diagnostics, Santa Maria, CA), nutrient agar (NA) (CriterionTM ; Hardy Diagnostics) and modified Vogel and Bonner’s medium (MVBM), which was a chemically defined medium used to facilitate the formation of biofilm [18]. Burkholderia pseudomallei 1026b was cultured aerobically in BHI broth at 37 ◦ C overnight and was then subcultured at 37 ◦ C in a 200 rpm shakerincubator for 1.5 h to yield a mid logarithmic growth phase. For all experiments except the antibiofilm assay, cells were re-suspended to the densities as indicated below. For the antibiofilm assay, a single colony of B. pseudomallei 1026b, H777 or M10 initially grown on LB agar or LB agar containing 15 g/mL tetracycline (for mutants)
was inoculated into 10 mL of MVBM and incubated at 37 ◦ C, 200 rpm for 16 h. Cells were re-suspended to the densities as indicated below. 2.3. Antibacterial assay The killing activities of all peptides against B. pseudomallei 1026b were determined by colony culturing assay as described previously [12]. Briefly, bacterial cells were washed three times and were re-suspended [ca. 107 colony-forming units (CFU)/mL] in 1 mM potassium phosphate buffer (PPB), pH 7.0. The bacterial suspension was then added to an equal volume of AMP to reach a final concentration of 100 M. A bacterial suspension in PPB without peptide served as a control. Following incubation at 37 ◦ C for 60 min, the incubation mixture was serially diluted in a physiological concentration of saline and plated in triplicate on NA. Colonies were counted after 24 h of incubation at 37 ◦ C. The percentage killing or inhibiting effects of each AMP was calculated using the formula [1 − (CFU sample/CFU control)] × 100%. Each assay was performed on two separate occasions, with duplicate determinations each time. 2.4. Killing kinetic assay of LL-37 and its most effective truncated variants Killing kinetics was determined using a culture of B. pseudomallei 1026b re-suspended in 1 mM PPB (ca. 105 CFU/mL). Each peptide was added to the bacterial suspension to a final concentration of 5, 10, 15 and 20 M and was incubated in a 200 rpm shaker-incubator at 37 ◦ C. At the indicated times (0, 1, 2, 4, 6 and 24 h), samples were taken, serially diluted, plated in triplicate on NA and incubated for 24 h to allow colony counting. The percentage killing or inhibiting effects of each AMP was calculated using the formula [1 − (CFU sample/CFU control)] × 100%. A bactericidal effect was defined as a ≥3 log10 reduction in CFU/mL compared with the initial inoculum. 2.5. Freeze-fracture electron microscopy Burkholderia pseudomallei 1026b (ca. 108 CFU/mL) in PPB was incubated with 20 M peptide for 1 h, collected by centrifugation and re-suspended in fixative solution (2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). Freeze-fracture electron microscopy was performed as described previously [19]. Replicas were examined with a transmission electron microscope (Philips EM-420; Philips, Eindhoven, The Netherlands) operated at 100 kV and equipped with a SIS MegaView II camera (Olympus Soft Imaging Solutions GmbH, Münster, Germany). 2.6. Release of nucleotides Burkholderia pseudomallei 1026b (ca. 108 CFU/mL) was incubated with 20 M peptide for 1 h and 2 h. At several time points, samples were collected and centrifuged at 5000 × g for 5 min. Release of the nucleotides AMP, ADP and ATP was analysed by capillary zone electrophoresis with a BioFocus 2000 Capillary Electrophoresis System (Bio-Rad Laboratories, Hercules, CA) equipped with an uncoated fused silica capillary of internal diameter 50 m and length 50 cm. Samples were monitored continuously at 260 nm. Peaks were quantified against the nucleotide standard containing a mixture of 10 M AMP, ADP and ATP [19]. 2.7. Antibiofilm assay Burkholderia pseudomallei 1026b, H777 and M10 were grown as biofilm using a modification of the Calgary Biofilm Device. This method of biofilm culture has been previously validated and
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Fig. 1. Antimicrobial activity of a library of LL-37 fragments with overlapping sequences against Burkholderia pseudomallei 1026b. Bacterial suspensions were incubated with 100 M peptides for 1 h and the viability of bacterial cells was determined by the plate count technique. Data are presented as the mean and standard deviation of two independent experiments performed in duplicate.
shown to be reproducible [20]. Each isolate (ca. 107 CFU/mL) was placed into a 96-well microtitre plate, then a transferable solidphase (TSP) pin lid (NUNC, Roskilde, Denmark) was placed into the microtitre plate, which was incubated in an orbital incubator at 37 ◦ C, 150 rpm, for 24 h. Following the period of incubation, biofilms were rinsed by inserting the TSP pin lids into microtitre plates containing 200 L/well of saline for 1 min to remove loosely adherent cells. The TSP pin lid with grown biofilms was placed into a new 96-well microtitre plate containing LL-31, LL-37 (20, 50 and 100 M) and CAZ (20, 50, 100, 936 and 1873 M) and then incubated at 37 ◦ C for 24 h. The concentrations of CAZ at 936 M (512 g/mL) and 1873 M (1024 g/mL) were included based on our previous study that these concentrations of CAZ were the minimal biofilm elimination concentration against B. pseudomallei 1026b, H777 and M10 [6]. Wells without antimicrobial agent were also included to control for growth due to the addition of media. After that, the TSP pin lid was removed again and rinsed with saline in another microtitre plate. After rinsing, the TSP pin lid was placed onto the new microtitre plate containing Muller–Hinton broth (recovery plate) and biofilms were disrupted from the TSP pin surface using an ultrasonic cleaner (SONOREX; Bandelin, Berlin, Germany) for 5 min. Viability of the biofilm bacteria was determined by plate counts. Colonies were counted after 24 h incubation at 37 ◦ C. The percentage killing effects of each concentration of LL-31, LL-37 and CAZ was calculated using the formula [1 − (CFU sample/CFU control)] × 100%. Each assay was performed on three separate occasions, with duplicate determinations each time.
3. Results 3.1. Antimicrobial activities of LL-37 and its truncated variants against Burkholderia pseudomallei 1026b The killing effect of peptides against B. pseudomallei 1026b is shown in Fig. 1. LL-37 and LL-31 exhibited 99.98% and 99.96% killing activities, respectively, whilst truncation of LL-37 from the C-terminus by 12 residues (LL-25) resulted in a >50% reduction of bactericidal activity. Further truncation completely abolished the bactericidal activity of the resulting peptides (LL-19 and LL13). In addition, truncation of LL-37 from the N-terminus (RK-31, IG-25, RI-19 and KD-13) also reduced bactericidal activity of the
peptides. However, truncation at both sides, from the N-terminus by 6 and 12 residues and the C-terminus by 6 residues (RK-25 and IG-19), restored the killing activities of both peptides to 99.4% and 99.59%, respectively, whilst >90% reduction of bactericidal activities occurred with further truncation of RK-25 and IG-19 from the C-terminus by six residues (RK-19 and IG-13). LL-31, the most effective truncated variant of LL-37, was selected to determine long-term killing kinetics in comparison with the native LL-37. The results showed that 20 M LL-31 killed all 105 CFU/mL bacterial cells within 1 h (Fig. 2B), whilst LL-37 showed complete killing within 2 h at the same concentration (Fig. 2A). Other concentrations of both peptides (5–15 M) showed initial reduction in CFU/mL in a dose-dependent manner, however they never reached a bactericidal endpoint and B. pseudomallei grew to ca. 104 CFU/mL after 24 h. 3.2. Ultrastructural membrane effects and membrane permeability The effects of LL-31 and the parent LL-37 on the membrane morphology of B. pseudomallei 1026b were determined by freezefracture electron microscopy. With this technique, bacterial cell fracturing occurs along the plane of hydrophobic interface between the inner and outer leaflets of the membrane. Untreated cells showed a normal inner and outer leaflet (Fig. 3A). However, irregularities and blebs were observed on the cell membranes after treatment with 20 M LL-31 and LL-37 (Fig. 3B and C). The membrane-permeabilising effects of both peptides were further demonstrated by measuring leakage of intracellular compounds such as nucleotides (AMP, ADP and ATP) from peptidetreated B. pseudomallei. Following treatment with LL-31 and LL-37 for 1 h and 2 h, a strong decrease in nucleotide content was detected within the bacterial cells compared with untreated bacteria (Fig. 4). 3.3. Antibiofilm activities of LL-37 and its most effective truncated variants compared with ceftazidime Three isolates of B. pseudomallei (1026b, the biofilm-defective mutant M10 and its wild-type H777) were selected to determine the antibiofilm activities of LL-31 and LL-37 compared with CAZ. All tested agents affected a clear dose- and strain-dependent killing activity against B. pseudomallei biofilms (Fig. 5). All concentrations of CAZ exhibited a smaller antibiofilm effect than the
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Fig. 2. Long-term killing kinetics of (A) LL-37 and (B) LL-31 against Burkholderia pseudomallei 1026b. Bacterial suspensions were incubated with the peptides (5–20 M) and samples were taken at the indicated time points (0, 1, 2, 4, 6 and 24 h). Colonies were counted and a bactericidal effect was defined as a ≥3 log10 reduction in colony-forming units (CFU)/mL compared with the initial inoculum. Data are the mean value of two independent experiments performed in duplicate.
Fig. 3. Freeze-fracture electron micrographs of Burkholderia pseudomallei 1026b cells: (A) B. pseudomallei incubated with potassium phosphate buffer (PPB) showed normal inner (black arrow head) and outer leaflets (white arrow head); (B) bacteria incubated with 20 M LL-31 showed irregularities and blebs (black arrows) on the membranes; (C) cells incubated with 20 M LL-37 demonstrated clearly irregular membranes and shapes of bacteria distinct from untreated bacteria. Scale bar, 500 nm.
minimum concentration used of both AMPs. The average percentage killing activities of CAZ at a concentration of 936 M (512 g/mL) and 1873 M (1024 g/mL) was <50% against all B. pseudomallei biofilms. In contrast, 20 M LL-31 exhibited 86%, 87% and 92% killing activities against biofilms of B. pseudomallei 1026b, H777 and M10, respectively, and >98% killing activities was
observed for all B. pseudomallei biofilms at 100 M LL-31. At the same concentrations, LL-37 showed lower killing activities against biofilms of all B. pseudomallei isolates tested. These results emphasise that LL-31 was the most effective fragment of LL-37 both against planktonic and biofilm forms of B. pseudomallei and possessed stronger antibiofilm activity than CAZ.
Fig. 4. Peptide-induced leakage of nucleotides from Burkholderia pseudomallei 1026b. Bacteria were incubated with 20 M peptides for 1 h and 2 h. Concentrations of nucleotides in the lysis fractions were measured by capillary zone electrophoresis. Data are presented as the mean and standard deviation of two independent experiments.
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Fig. 5. Antimicrobial activity of LL-37 and LL-31 against Burkholderia pseudomallei in biofilm compared with ceftazidime. The biofilm was incubated with several concentrations of peptides and ceftazidime for 24 h and the viability of bacterial cells was determined by the plate count technique. Data are presented as the mean and standard deviation of two independent experiments performed in duplicate.
4. Discussion In this study, it was found that the shortest fragment of LL-37 that possessed the strongest killing activity against B. pseudomallei was LL-31, similar to our previous observations against B. thailandensis [13]. Moreover, B. pseudomallei responds the same as B. thailandensis, with IG-19 as the smallest active peptide. These results clearly indicated that the sequence IGKEFKRIVQRIKDFLRNL (residues 13–31) forms the minimal domain necessary for bactericidal activity both against B. thailandensis E264 and B. pseudomallei 1026b. Obviously, the sequence VPRTES (residues 31–37) at the C-terminal is not crucial for antimicrobial activity, whereas the sequence DFLRNL (residues 26–31) is more important for antimicrobial potency because the derivatives that lack this sequence (e.g. LL-13, LL-19, LL-25, RK-19 and IG-13) had less to no killing effect. Truncation of LL-37 from the N-terminus by six residues (RK-31) resulted in a decreased bactericidal activity compared with LL-37 and LL-31 but still exhibited a strong killing effect, whereas a strong reduction of bactericidal activity of RK-31 was observed towards B. thailandensis [13]. Previous analysis of the structurally distinct cathelicidin PR-39 has shown that N-terminal lysines are important to activity and may function by facilitating initial ionic interaction with the anionic microbial surface [21]. This may explain the reduction in bactericidal activity observed in the present study after cleavage of the amino acids at the N-terminus of LL-37 (RK-31, IG-25, RI-19 and KD-13). However, this explanation is probably incomplete because truncation at both sides (RK-25 and IG-19) restored the bactericidal activity. Moreover, a gain in antimicrobial activity after processing of LL-37 to RK-31 was observed also against Staphylococcus aureus, Escherichia coli and Candida albicans [22,23]. Truncation of peptides by deletion of amino acids has different effects on several physicochemical properties that together determine the antimicrobial activity. The percentage ␣-helical structure of LL-37 as determined by circular dichroism experiments remains similar after truncation to LL-31 [24]. Nevertheless, there is no strict correlation between the observed Burkholderia killing activity and the amount of ␣-helical structure; for instance, IG-19 is as active as LL-37 and LL-31 (Fig. 1), whilst the propensity to adopt an ␣-helix has been shown to be only one-half that of LL37 [24]. The other way round, LL-25, which has a percentage of ␣-helicity similar to LL-37, shows only 30% of the killing activity of LL-37.
Freeze-fracture electron microscopy of bacterial cells demonstrated that LL-31 disrupted the membrane both of B. pseudomallei and B. thailandensis [13] and caused leakage of intracellular molecules leading to cell death. However, the results from longterm killing kinetics of LL-31 revealed that B. pseudomallei was less susceptible than B. thailandensis because LL-31 at a concentration of 5 M killed all 105 CFU/mL B. thailandensis within 6 h [13]. In contrast, 5–15 M LL-31 never reached a bactericidal endpoint and B. pseudomallei grew to ca. 104 CFU/mL after 24 h. These results suggested that the membrane structure and membrane biochemistry of B. pseudomallei and B. thailandensis may be different, although B. thailandensis displays several genetic and phenotypic features that are very similar to B. pseudomallei [25]. Recently, structural analysis of B. pseudomallei lipopolysaccharide (LPS) revealed that both phosphate groups in the major lipid A species identified in B. pseudomallei are capped with 4-amino-4-deoxy-arabinose (Ara4N) residues, which was different from B. thailandensis LPS [26]. Ara4N lipid A modifications reduce the negative charge of the bacterial membrane resulting in increased bacterial resistance to cationic AMPs and cationic antibiotics by altering the binding of these peptides to the membrane [27,28]. Therefore, these findings may explain the lower susceptibility of B. pseudomallei to LL-37 and LL-31 found in this study compared with B. thailandensis in our previous study [13]. Burkholderia pseudomallei has been reported to form biofilm both in vitro and in vivo [5]. We have demonstrated that pregrown B. pseudomallei biofilms were markedly resistant to all antimicrobial agents tested (doxycycline, CAZ, imipenem and trimethoprim/sulfamethoxazole) compared with the corresponding planktonic cells of the same isolates [6]. It is well accepted that bacteria growing in a biofilm are more recalcitrant to the action of antibiotics than cells growing in a planktonic state [29]. One proposed mechanism of the resistance is the slow growth rate and low metabolic activity of bacteria in biofilms, because most antimicrobial agents, particularly -lactams, primarily target metabolically active cells [30,31]. In contrast, AMPs have a high potential to act on slow-growing or even non-growing bacteria because they have the ability to permeabilise and/or to form pores within the cytoplasmic membrane. In the present study, it was found that LL-37 and LL-31 exhibited a stronger antibiofilm than CAZ, in line with the above proposed mechanism of action of these peptides. In addition, amongst three isolates of B. pseudomallei, the biofilm-defective
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mutant M10 was the most susceptible to LL-37 and LL-31. This may be due to the different amount of extracellular polymeric substance matrix that embedded the cells. In conclusion, these results show that LL-31 was the most effective peptide against B. pseudomallei both in planktonic and biofilm form; therefore it should be considered for the development as a new potential therapeutic agent that may help in the treatment of melioidosis. Funding: This work was supported by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Health Cluster (SHep-GMS), Khon Kaen University. JGMB, ECIV, KN and HJJdS are supported by a grant from the University of Amsterdam for research into the focal point ‘Oral infections and inflammation’. ECIV greatly acknowledges support from the Skeletal Tissue Engineering Group Amsterdam (STEGA). Competing interests: None declared. Ethical approval: Not required. References [1] Cheng AC, Currie BJ, Melioidosis:. epidemiology, pathophysiology, and management. Clin Microbiol Rev 2005;18:383–416. [2] White NJ, Melioidosis. Lancet 2003;361:1715–22. [3] Howe C, Sampath A, Spotnitz M. The pseudomallei group: a review. J Infect Dis 1971;124:598–606. [4] Leelarasamee A, Bovornkitti S. Melioidosis: review and update. Rev Infect Dis 1989;11:413–25. [5] Vorachit M, Lam K, Jayanetra P, Costerton JW. Electron microscopy study of the mode of growth of Pseudomonas pseudomallei in vitro and in vivo. J Trop Med Hyg 1995;98:379–91. [6] Sawasdidoln C, Taweechaisupapong S, Sermswan RW, Tattawasart U, Tungpradabkul S, Wongratanacheewin S. Growing Burkholderia pseudomallei in biofilm stimulating conditions significantly induces antimicrobial resistance. PLoS One 2010;5, e9196. [7] Larrick JW, Hirata M, Zhong J, Wright SC. Anti-microbial activity of human CAP18 peptides. Immunotechnology 1995;1:65–72. [8] Travis SM, Anderson NN, Forsyth WR, Espiritu C, Conway BD, Greenberg EP, et al. Bactericidal activity of mammalian cathelicidin-derived peptides. Infect Immun 2000;68:2748–55. [9] Turner J, Cho Y, Dinh NN, Waring AJ, Lehrer RI. Activities of LL-37, a cathelinassociated antimicrobial peptide of human neutrophils. Antimicrob Agents Chemother 1998;42:2206–14. [10] Hell E, Giske CG, Nelson A, Romling U, Marchini G. Human cathelicidin peptide LL37 inhibits both attachment capability and biofilm formation of Staphylococcus epidermidis. Lett Appl Microbiol 2010;50:211–5. [11] Overhage J, Campisano A, Bains M, Torfs EC, Rehm BH, Hancock RE. Human host defense peptide LL-37 prevents bacterial biofilm formation. Infect Immun 2008;76:4176–82. [12] Kanthawong S, Nazmi K, Wongratanacheewin S, Bolscher JG, Wuthiekanun V, Taweechaisupapong S. In vitro susceptibility of Burkholderia pseudomallei to antimicrobial peptides. Int J Antimicrob Agents 2009;34: 309–14.
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International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Antimicrobial and antibiofilm activity of LL-37 and its truncated variants against Burkholderia pseudomallei Sakawrat Kanthawong a,b , Jan G.M. Bolscher c , Enno C.I. Veerman c , Jan van Marle d , Hans J.J. de Soet e , Kamran Nazmi c , Surasakdi Wongratanacheewin a,b , Suwimol Taweechaisupapong b,f,∗ a
Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Melioidosis Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand c Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, 1081 BT Amsterdam, The Netherlands d Department of Electron Microscopy, Academic Medical Center, University of Amsterdam, The Netherlands e Department of Oral Microbiology, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, 1081 BT Amsterdam, The Netherlands f Biofilm Research Group, Department of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand b
a r t i c l e
i n f o
Article history: Received 24 June 2011 Accepted 9 September 2011 Keywords: Burkholderia pseudomallei Antimicrobial peptide LL-37 Truncated variants Biofilm
a b s t r a c t The Gram-negative bacterium Burkholderia pseudomallei is the aetiological agent of melioidosis, which is an endemic disease in tropical areas of Southeast Asia and Northern Australia. Burkholderia pseudomallei has intrinsic resistance to a number of commonly used antibiotics and has also been reported to develop a biofilm. Resistance to killing by antimicrobial agents is one of the hallmarks of bacteria grown in biofilm. The aim of this study was to determine the antimicrobial activity and mechanisms of action of LL-37 and its truncated variants against B. pseudomallei both in planktonic and biofilm form, as LL-37 is an antimicrobial peptide that possessed strong killing activity against several pathogens. Antimicrobial assays revealed that LL-31, a truncated variant of LL-37 lacking the six C-terminus residues, exhibited the strongest killing effect. Time–kill experiments showed that 20 M LL-31 can reach the bactericidal endpoint within 2 h. Freeze-fracture electron microscopy of bacterial cells demonstrated that these peptides disrupt the membrane and cause leakage of intracellular molecules leading to cell death. Moreover, LL-31 also possessed stronger bactericidal activity than ceftazidime against B. pseudomallei grown in biofilm. Thus, LL-31 should be considered as a potent antimicrobial agent against B. pseudomallei both in planktonic and biofilm form. © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Burkholderia pseudomallei is the causative agent of melioidosis, a life-threatening bacterial infectious disease of man and animals in Southeast Asia, Northern Australia, the Indian subcontinent, Iran, and Central and South America [1,2]. Clinical manifestations range from seropositive subclinical infection to acute fatal septicaemia or chronic disease [2]. Infection is thought to be acquired either through a wound in the skin or through inhalation of aerosolised B. pseudomallei. Successful treatment of melioidosis patients is difficult because the pathogen is inherently resistant to a variety of antibiotics, including -lactams, aminoglycosides, macrolides and polymyxins [3,4]. Ceftazidime (CAZ), carbapenems such as imipenem and meropenem, and, to a lesser degree,
∗ Corresponding author. Present address: Biofilm Research Group, Department of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand. Fax: +66 43 202 862. E-mail address: suvi [email protected] (S. Taweechaisupapong).
amoxicillin/clavulanic acid remain the backbone of current initial or intensive-phase melioidosis treatment. Resistance to CAZ and imipenem is rare. The current standard treatment with agents to which B. pseudomallei is susceptible requires 2–4 weeks of parenteral therapy, e.g. with CAZ as initial intensive therapy, followed by 3–6 months of oral eradication therapy, e.g. with trimethoprim/sulfamethoxazole, doxycycline, chloramphenicol or a combination therapy. Although CAZ is the drug of choice that is the most effective for treatment of severe melioidosis, the mortality rate in treated patients is >40% [2]. In addition, B. pseudomallei was reported to form biofilm and microcolonies [5]. Burkholderia pseudomallei growing in biofilm is resistant to several conventional antibiotics [6]. This may be proposed as the cause of relapse cases in melioidosis patients. With the increasing development of antibiotic resistance, it is worth searching for novel anti-infective agents against B. pseudomallei. LL-37, the only human member of the cathelicidin family of antimicrobial peptides (AMPs), has killing activity against a broad range of Gram-positive and Gram-negative bacteria in vitro [7–9]. The inhibitory effects of LL-37 on biofilm formation as well as
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the initial attachment of Staphylococcus epidermidis and Pseudomonas aeruginosa have been reported [10,11]. Recently, we found that LL-37 has strong killing activity against B. pseudomallei [12]. Further investigation of a library of LL-37 fragments with overlapping sequences using Burkholderia thailandensis E264 as a model demonstrated that LL-31, a truncated variant of LL-37 missing the six residues at the C-terminus, displayed the strongest killing effect against B. thailandensis E264 by disrupting the bacterial membrane and subsequent leakage of intracellular components leading to death of the bacterium [13]. Although B. thailandensis is closely related to B. pseudomallei [14], susceptibility to antimicrobial agents differs [15]. Therefore, in the present study, a library of LL-37 fragments was further investigated with B. pseudomallei both in planktonic and biofilm form in comparison with the conventional antibiotic CAZ. 2. Materials and methods 2.1. Peptide synthesis, purification and labelling The human cathelicidin peptide LL-37 and its truncated variants [LL-31, LL-25, LL-19, LL-13, RK-31 (LL 7-37), IG-25 (LL 13-37), RI-19 (LL 19-37), KD-13 (LL 25-37), RK-25 (LL 7-31), IG-19 (LL 13-31), RK-19 (LL 9-25) and IG-13 (LL 13-25)] were synthesised by fluoren-9-ylmethoxycarbonyl (Fmoc) chemistry using a MilliGen 9050 Peptide Synthesizer (MilliGen/BioSearch, Bedford, MA) as described previously [16]. For the fluorescein isothiocyanate (FITC) labelling, peptides were extended with the linker FmocL-␥-aminobutyric acid and, after detachment of the Fmoc group, were labelled overnight at 20 ◦ C with 30-fold excess of FITC in N,Ndiisopropylethylamine/dimethylformamide (DIPEA/DMF) before removal of the side chain protecting groups and simultaneous detachment from the resin support. All peptides were of ≥95% purity as determined by reverse-phase high-performance liquid chromatography (RP-HPLC) (JASCO, Tokyo, Japan). The sequences of all peptides investigated are shown in Fig. 1. 2.2. Bacterial strains and growth conditions Burkholderia pseudomallei isolate 1026b as well as H777 (wildtype) and a biofilm-defective mutant (M10) [17] were used in this study. Burkholderia pseudomallei isolate 1026b was kindly provided by Dr W.J. Wiersinga [Center for Infection and Immunity (CINIMA), Center for Experimental and Molecular Medicine, University of Amsterdam, Academic Medical Centre, Amsterdam, The Netherlands] with permission of Prof. Dr D.E. Woods (Department of Microbiology and Infectious Diseases, University of Calgary Health Science Center, Calgary, Alberta, Canada). Burkholderia pseudomallei isolate H777 was isolated from a patient admitted to Srinagarind Hospital (Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand). Media used in this study were brain–heart infusion (BHI) broth (Difco, Becton Dickinson Microbiology Systems, Franklin Lakes, NJ), Luria–Bertani (LB) agar (Difco), Muller–Hinton broth (CriterionTM ; Hardy Diagnostics, Santa Maria, CA), nutrient agar (NA) (CriterionTM ; Hardy Diagnostics) and modified Vogel and Bonner’s medium (MVBM), which was a chemically defined medium used to facilitate the formation of biofilm [18]. Burkholderia pseudomallei 1026b was cultured aerobically in BHI broth at 37 ◦ C overnight and was then subcultured at 37 ◦ C in a 200 rpm shakerincubator for 1.5 h to yield a mid logarithmic growth phase. For all experiments except the antibiofilm assay, cells were re-suspended to the densities as indicated below. For the antibiofilm assay, a single colony of B. pseudomallei 1026b, H777 or M10 initially grown on LB agar or LB agar containing 15 g/mL tetracycline (for mutants)
was inoculated into 10 mL of MVBM and incubated at 37 ◦ C, 200 rpm for 16 h. Cells were re-suspended to the densities as indicated below. 2.3. Antibacterial assay The killing activities of all peptides against B. pseudomallei 1026b were determined by colony culturing assay as described previously [12]. Briefly, bacterial cells were washed three times and were re-suspended [ca. 107 colony-forming units (CFU)/mL] in 1 mM potassium phosphate buffer (PPB), pH 7.0. The bacterial suspension was then added to an equal volume of AMP to reach a final concentration of 100 M. A bacterial suspension in PPB without peptide served as a control. Following incubation at 37 ◦ C for 60 min, the incubation mixture was serially diluted in a physiological concentration of saline and plated in triplicate on NA. Colonies were counted after 24 h of incubation at 37 ◦ C. The percentage killing or inhibiting effects of each AMP was calculated using the formula [1 − (CFU sample/CFU control)] × 100%. Each assay was performed on two separate occasions, with duplicate determinations each time. 2.4. Killing kinetic assay of LL-37 and its most effective truncated variants Killing kinetics was determined using a culture of B. pseudomallei 1026b re-suspended in 1 mM PPB (ca. 105 CFU/mL). Each peptide was added to the bacterial suspension to a final concentration of 5, 10, 15 and 20 M and was incubated in a 200 rpm shaker-incubator at 37 ◦ C. At the indicated times (0, 1, 2, 4, 6 and 24 h), samples were taken, serially diluted, plated in triplicate on NA and incubated for 24 h to allow colony counting. The percentage killing or inhibiting effects of each AMP was calculated using the formula [1 − (CFU sample/CFU control)] × 100%. A bactericidal effect was defined as a ≥3 log10 reduction in CFU/mL compared with the initial inoculum. 2.5. Freeze-fracture electron microscopy Burkholderia pseudomallei 1026b (ca. 108 CFU/mL) in PPB was incubated with 20 M peptide for 1 h, collected by centrifugation and re-suspended in fixative solution (2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). Freeze-fracture electron microscopy was performed as described previously [19]. Replicas were examined with a transmission electron microscope (Philips EM-420; Philips, Eindhoven, The Netherlands) operated at 100 kV and equipped with a SIS MegaView II camera (Olympus Soft Imaging Solutions GmbH, Münster, Germany). 2.6. Release of nucleotides Burkholderia pseudomallei 1026b (ca. 108 CFU/mL) was incubated with 20 M peptide for 1 h and 2 h. At several time points, samples were collected and centrifuged at 5000 × g for 5 min. Release of the nucleotides AMP, ADP and ATP was analysed by capillary zone electrophoresis with a BioFocus 2000 Capillary Electrophoresis System (Bio-Rad Laboratories, Hercules, CA) equipped with an uncoated fused silica capillary of internal diameter 50 m and length 50 cm. Samples were monitored continuously at 260 nm. Peaks were quantified against the nucleotide standard containing a mixture of 10 M AMP, ADP and ATP [19]. 2.7. Antibiofilm assay Burkholderia pseudomallei 1026b, H777 and M10 were grown as biofilm using a modification of the Calgary Biofilm Device. This method of biofilm culture has been previously validated and
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Fig. 1. Antimicrobial activity of a library of LL-37 fragments with overlapping sequences against Burkholderia pseudomallei 1026b. Bacterial suspensions were incubated with 100 M peptides for 1 h and the viability of bacterial cells was determined by the plate count technique. Data are presented as the mean and standard deviation of two independent experiments performed in duplicate.
shown to be reproducible [20]. Each isolate (ca. 107 CFU/mL) was placed into a 96-well microtitre plate, then a transferable solidphase (TSP) pin lid (NUNC, Roskilde, Denmark) was placed into the microtitre plate, which was incubated in an orbital incubator at 37 ◦ C, 150 rpm, for 24 h. Following the period of incubation, biofilms were rinsed by inserting the TSP pin lids into microtitre plates containing 200 L/well of saline for 1 min to remove loosely adherent cells. The TSP pin lid with grown biofilms was placed into a new 96-well microtitre plate containing LL-31, LL-37 (20, 50 and 100 M) and CAZ (20, 50, 100, 936 and 1873 M) and then incubated at 37 ◦ C for 24 h. The concentrations of CAZ at 936 M (512 g/mL) and 1873 M (1024 g/mL) were included based on our previous study that these concentrations of CAZ were the minimal biofilm elimination concentration against B. pseudomallei 1026b, H777 and M10 [6]. Wells without antimicrobial agent were also included to control for growth due to the addition of media. After that, the TSP pin lid was removed again and rinsed with saline in another microtitre plate. After rinsing, the TSP pin lid was placed onto the new microtitre plate containing Muller–Hinton broth (recovery plate) and biofilms were disrupted from the TSP pin surface using an ultrasonic cleaner (SONOREX; Bandelin, Berlin, Germany) for 5 min. Viability of the biofilm bacteria was determined by plate counts. Colonies were counted after 24 h incubation at 37 ◦ C. The percentage killing effects of each concentration of LL-31, LL-37 and CAZ was calculated using the formula [1 − (CFU sample/CFU control)] × 100%. Each assay was performed on three separate occasions, with duplicate determinations each time.
3. Results 3.1. Antimicrobial activities of LL-37 and its truncated variants against Burkholderia pseudomallei 1026b The killing effect of peptides against B. pseudomallei 1026b is shown in Fig. 1. LL-37 and LL-31 exhibited 99.98% and 99.96% killing activities, respectively, whilst truncation of LL-37 from the C-terminus by 12 residues (LL-25) resulted in a >50% reduction of bactericidal activity. Further truncation completely abolished the bactericidal activity of the resulting peptides (LL-19 and LL13). In addition, truncation of LL-37 from the N-terminus (RK-31, IG-25, RI-19 and KD-13) also reduced bactericidal activity of the
peptides. However, truncation at both sides, from the N-terminus by 6 and 12 residues and the C-terminus by 6 residues (RK-25 and IG-19), restored the killing activities of both peptides to 99.4% and 99.59%, respectively, whilst >90% reduction of bactericidal activities occurred with further truncation of RK-25 and IG-19 from the C-terminus by six residues (RK-19 and IG-13). LL-31, the most effective truncated variant of LL-37, was selected to determine long-term killing kinetics in comparison with the native LL-37. The results showed that 20 M LL-31 killed all 105 CFU/mL bacterial cells within 1 h (Fig. 2B), whilst LL-37 showed complete killing within 2 h at the same concentration (Fig. 2A). Other concentrations of both peptides (5–15 M) showed initial reduction in CFU/mL in a dose-dependent manner, however they never reached a bactericidal endpoint and B. pseudomallei grew to ca. 104 CFU/mL after 24 h. 3.2. Ultrastructural membrane effects and membrane permeability The effects of LL-31 and the parent LL-37 on the membrane morphology of B. pseudomallei 1026b were determined by freezefracture electron microscopy. With this technique, bacterial cell fracturing occurs along the plane of hydrophobic interface between the inner and outer leaflets of the membrane. Untreated cells showed a normal inner and outer leaflet (Fig. 3A). However, irregularities and blebs were observed on the cell membranes after treatment with 20 M LL-31 and LL-37 (Fig. 3B and C). The membrane-permeabilising effects of both peptides were further demonstrated by measuring leakage of intracellular compounds such as nucleotides (AMP, ADP and ATP) from peptidetreated B. pseudomallei. Following treatment with LL-31 and LL-37 for 1 h and 2 h, a strong decrease in nucleotide content was detected within the bacterial cells compared with untreated bacteria (Fig. 4). 3.3. Antibiofilm activities of LL-37 and its most effective truncated variants compared with ceftazidime Three isolates of B. pseudomallei (1026b, the biofilm-defective mutant M10 and its wild-type H777) were selected to determine the antibiofilm activities of LL-31 and LL-37 compared with CAZ. All tested agents affected a clear dose- and strain-dependent killing activity against B. pseudomallei biofilms (Fig. 5). All concentrations of CAZ exhibited a smaller antibiofilm effect than the
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Fig. 2. Long-term killing kinetics of (A) LL-37 and (B) LL-31 against Burkholderia pseudomallei 1026b. Bacterial suspensions were incubated with the peptides (5–20 M) and samples were taken at the indicated time points (0, 1, 2, 4, 6 and 24 h). Colonies were counted and a bactericidal effect was defined as a ≥3 log10 reduction in colony-forming units (CFU)/mL compared with the initial inoculum. Data are the mean value of two independent experiments performed in duplicate.
Fig. 3. Freeze-fracture electron micrographs of Burkholderia pseudomallei 1026b cells: (A) B. pseudomallei incubated with potassium phosphate buffer (PPB) showed normal inner (black arrow head) and outer leaflets (white arrow head); (B) bacteria incubated with 20 M LL-31 showed irregularities and blebs (black arrows) on the membranes; (C) cells incubated with 20 M LL-37 demonstrated clearly irregular membranes and shapes of bacteria distinct from untreated bacteria. Scale bar, 500 nm.
minimum concentration used of both AMPs. The average percentage killing activities of CAZ at a concentration of 936 M (512 g/mL) and 1873 M (1024 g/mL) was <50% against all B. pseudomallei biofilms. In contrast, 20 M LL-31 exhibited 86%, 87% and 92% killing activities against biofilms of B. pseudomallei 1026b, H777 and M10, respectively, and >98% killing activities was
observed for all B. pseudomallei biofilms at 100 M LL-31. At the same concentrations, LL-37 showed lower killing activities against biofilms of all B. pseudomallei isolates tested. These results emphasise that LL-31 was the most effective fragment of LL-37 both against planktonic and biofilm forms of B. pseudomallei and possessed stronger antibiofilm activity than CAZ.
Fig. 4. Peptide-induced leakage of nucleotides from Burkholderia pseudomallei 1026b. Bacteria were incubated with 20 M peptides for 1 h and 2 h. Concentrations of nucleotides in the lysis fractions were measured by capillary zone electrophoresis. Data are presented as the mean and standard deviation of two independent experiments.
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Fig. 5. Antimicrobial activity of LL-37 and LL-31 against Burkholderia pseudomallei in biofilm compared with ceftazidime. The biofilm was incubated with several concentrations of peptides and ceftazidime for 24 h and the viability of bacterial cells was determined by the plate count technique. Data are presented as the mean and standard deviation of two independent experiments performed in duplicate.
4. Discussion In this study, it was found that the shortest fragment of LL-37 that possessed the strongest killing activity against B. pseudomallei was LL-31, similar to our previous observations against B. thailandensis [13]. Moreover, B. pseudomallei responds the same as B. thailandensis, with IG-19 as the smallest active peptide. These results clearly indicated that the sequence IGKEFKRIVQRIKDFLRNL (residues 13–31) forms the minimal domain necessary for bactericidal activity both against B. thailandensis E264 and B. pseudomallei 1026b. Obviously, the sequence VPRTES (residues 31–37) at the C-terminal is not crucial for antimicrobial activity, whereas the sequence DFLRNL (residues 26–31) is more important for antimicrobial potency because the derivatives that lack this sequence (e.g. LL-13, LL-19, LL-25, RK-19 and IG-13) had less to no killing effect. Truncation of LL-37 from the N-terminus by six residues (RK-31) resulted in a decreased bactericidal activity compared with LL-37 and LL-31 but still exhibited a strong killing effect, whereas a strong reduction of bactericidal activity of RK-31 was observed towards B. thailandensis [13]. Previous analysis of the structurally distinct cathelicidin PR-39 has shown that N-terminal lysines are important to activity and may function by facilitating initial ionic interaction with the anionic microbial surface [21]. This may explain the reduction in bactericidal activity observed in the present study after cleavage of the amino acids at the N-terminus of LL-37 (RK-31, IG-25, RI-19 and KD-13). However, this explanation is probably incomplete because truncation at both sides (RK-25 and IG-19) restored the bactericidal activity. Moreover, a gain in antimicrobial activity after processing of LL-37 to RK-31 was observed also against Staphylococcus aureus, Escherichia coli and Candida albicans [22,23]. Truncation of peptides by deletion of amino acids has different effects on several physicochemical properties that together determine the antimicrobial activity. The percentage ␣-helical structure of LL-37 as determined by circular dichroism experiments remains similar after truncation to LL-31 [24]. Nevertheless, there is no strict correlation between the observed Burkholderia killing activity and the amount of ␣-helical structure; for instance, IG-19 is as active as LL-37 and LL-31 (Fig. 1), whilst the propensity to adopt an ␣-helix has been shown to be only one-half that of LL37 [24]. The other way round, LL-25, which has a percentage of ␣-helicity similar to LL-37, shows only 30% of the killing activity of LL-37.
Freeze-fracture electron microscopy of bacterial cells demonstrated that LL-31 disrupted the membrane both of B. pseudomallei and B. thailandensis [13] and caused leakage of intracellular molecules leading to cell death. However, the results from longterm killing kinetics of LL-31 revealed that B. pseudomallei was less susceptible than B. thailandensis because LL-31 at a concentration of 5 M killed all 105 CFU/mL B. thailandensis within 6 h [13]. In contrast, 5–15 M LL-31 never reached a bactericidal endpoint and B. pseudomallei grew to ca. 104 CFU/mL after 24 h. These results suggested that the membrane structure and membrane biochemistry of B. pseudomallei and B. thailandensis may be different, although B. thailandensis displays several genetic and phenotypic features that are very similar to B. pseudomallei [25]. Recently, structural analysis of B. pseudomallei lipopolysaccharide (LPS) revealed that both phosphate groups in the major lipid A species identified in B. pseudomallei are capped with 4-amino-4-deoxy-arabinose (Ara4N) residues, which was different from B. thailandensis LPS [26]. Ara4N lipid A modifications reduce the negative charge of the bacterial membrane resulting in increased bacterial resistance to cationic AMPs and cationic antibiotics by altering the binding of these peptides to the membrane [27,28]. Therefore, these findings may explain the lower susceptibility of B. pseudomallei to LL-37 and LL-31 found in this study compared with B. thailandensis in our previous study [13]. Burkholderia pseudomallei has been reported to form biofilm both in vitro and in vivo [5]. We have demonstrated that pregrown B. pseudomallei biofilms were markedly resistant to all antimicrobial agents tested (doxycycline, CAZ, imipenem and trimethoprim/sulfamethoxazole) compared with the corresponding planktonic cells of the same isolates [6]. It is well accepted that bacteria growing in a biofilm are more recalcitrant to the action of antibiotics than cells growing in a planktonic state [29]. One proposed mechanism of the resistance is the slow growth rate and low metabolic activity of bacteria in biofilms, because most antimicrobial agents, particularly -lactams, primarily target metabolically active cells [30,31]. In contrast, AMPs have a high potential to act on slow-growing or even non-growing bacteria because they have the ability to permeabilise and/or to form pores within the cytoplasmic membrane. In the present study, it was found that LL-37 and LL-31 exhibited a stronger antibiofilm than CAZ, in line with the above proposed mechanism of action of these peptides. In addition, amongst three isolates of B. pseudomallei, the biofilm-defective
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mutant M10 was the most susceptible to LL-37 and LL-31. This may be due to the different amount of extracellular polymeric substance matrix that embedded the cells. In conclusion, these results show that LL-31 was the most effective peptide against B. pseudomallei both in planktonic and biofilm form; therefore it should be considered for the development as a new potential therapeutic agent that may help in the treatment of melioidosis. Funding: This work was supported by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Health Cluster (SHep-GMS), Khon Kaen University. JGMB, ECIV, KN and HJJdS are supported by a grant from the University of Amsterdam for research into the focal point ‘Oral infections and inflammation’. ECIV greatly acknowledges support from the Skeletal Tissue Engineering Group Amsterdam (STEGA). Competing interests: None declared. Ethical approval: Not required. References [1] Cheng AC, Currie BJ, Melioidosis:. epidemiology, pathophysiology, and management. Clin Microbiol Rev 2005;18:383–416. [2] White NJ, Melioidosis. Lancet 2003;361:1715–22. [3] Howe C, Sampath A, Spotnitz M. The pseudomallei group: a review. J Infect Dis 1971;124:598–606. [4] Leelarasamee A, Bovornkitti S. Melioidosis: review and update. Rev Infect Dis 1989;11:413–25. [5] Vorachit M, Lam K, Jayanetra P, Costerton JW. Electron microscopy study of the mode of growth of Pseudomonas pseudomallei in vitro and in vivo. J Trop Med Hyg 1995;98:379–91. [6] Sawasdidoln C, Taweechaisupapong S, Sermswan RW, Tattawasart U, Tungpradabkul S, Wongratanacheewin S. Growing Burkholderia pseudomallei in biofilm stimulating conditions significantly induces antimicrobial resistance. PLoS One 2010;5, e9196. [7] Larrick JW, Hirata M, Zhong J, Wright SC. Anti-microbial activity of human CAP18 peptides. Immunotechnology 1995;1:65–72. [8] Travis SM, Anderson NN, Forsyth WR, Espiritu C, Conway BD, Greenberg EP, et al. Bactericidal activity of mammalian cathelicidin-derived peptides. Infect Immun 2000;68:2748–55. [9] Turner J, Cho Y, Dinh NN, Waring AJ, Lehrer RI. Activities of LL-37, a cathelinassociated antimicrobial peptide of human neutrophils. Antimicrob Agents Chemother 1998;42:2206–14. [10] Hell E, Giske CG, Nelson A, Romling U, Marchini G. Human cathelicidin peptide LL37 inhibits both attachment capability and biofilm formation of Staphylococcus epidermidis. Lett Appl Microbiol 2010;50:211–5. [11] Overhage J, Campisano A, Bains M, Torfs EC, Rehm BH, Hancock RE. Human host defense peptide LL-37 prevents bacterial biofilm formation. Infect Immun 2008;76:4176–82. [12] Kanthawong S, Nazmi K, Wongratanacheewin S, Bolscher JG, Wuthiekanun V, Taweechaisupapong S. In vitro susceptibility of Burkholderia pseudomallei to antimicrobial peptides. Int J Antimicrob Agents 2009;34: 309–14.
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