Membrane-active mechanism of LFchimera against Burkholderia pseudomallei and Burkholderia thailandensis.

LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265-284 and lactoferricin17-30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.

Chimerization of lactoferricin and lactoferrampin peptides strongly potentiates the killing activity against Candida albicans.

Bovine lactoferrin harbors 2 antimicrobial sequences (LFcin and LFampin), situated in close proximity in the N1-domain. To mimic their semi parallel configuration we have synthesized a chimeric peptide (LFchimera) in which these sequences are linked in a head-to-head fashion to the α- and ε-amino group, respectively, of a single lysine. In line with previously described bactericidal effects, this peptide was also a stronger candidacidal agent than the antimicrobial peptides LFcin17-30 and LFampin265-284, or a combination of these 2. Conditions that strongly reduced the candidacidal activities of LFcin17-30 and LFampin265-284, such as high ionic strength and energy depletion, had little influence on the activity of LFchimera. Freeze-fracture electron microscopy showed that LFchimera severely affected the membrane morphology, resulting in disintegration of the membrane bilayer and in an efflux of small and high molecular weight molecules such as ATP and proteins. The differential effects displayed by the chimeric peptide and a mixture of its constituent peptides clearly demonstrate the synergistic effect of linking these peptides in a fashion that allows a similar spatial arrangement as in the parent protein, suggesting that in bovine lactoferrrin the corresponding fragments act in concert in its candidacidal activity.

Hyposplenism: comparison of different methods for determining splenic function.

Asplenic patients are at risk for pneumococcal sepsis. Patients with hyposplenic function, such as associated with sickle cell disease (SCD), are also at risk. However, tests to assess splenic function are either unavailable or lacking standardization. The aim of this study was to compare different methods for determining splenic function. Eighteen patients with SCD (i.e., 10 heterozygous (SC) and 8 homozygous (SS) SCD patients), and eight splenectomized patients were compared to 10 controls. All subjects underwent spleen scintigraphy, after which functional splenic volumes (FSV) were calculated. FSV was compared to immunological function and B cell-subsets, as well as phagocytic function represented by the presence of Howell Jolly bodies (HJB) and percentages of pitted red cells (PIT). Heterozygous SCD (SC) patients had increased splenic volumes, but diminished FSV, homozygous SCD (SS) patients were asplenic. Splenectomized and SS patients had a strongly reduced phagocytic and immunological function. SC patients had reduced anti-polysaccharide responses without an increase in PIT. FSV correlated significantly with phagocytic and immunological function. HJB were indicative of splenic dysfunction, HJB absence was not indicative of normal functioning splenic tissue. Although visualizing HJB is methodologically advantageous to PIT, both are valid biomarkers of splenic dysfunction. The amount of non-switched memory B cells is strongly correlated to FSV.

Adaptation of the dermal collagen structure of human skin and scar tissue in response to stretch: an experimental study.

Surgeons are often faced with large defects that are difficult to close. Stretching adjacent skin can facilitate wound closure. In clinical practice, intraoperative stretching is performed in a cyclical or continuous fashion. However, exact mechanisms of tissue adaptation to stretch remain unclear. Therefore, we investigated collagen and elastin orientation and morphology of stretched and nonstretched healthy skin and scars. Tissue samples were stretched, fixed in stretched-out position, and processed for histology. Objective methods were used to quantify the collagen orientation index (COI), bundle thickness, and bundle spacing. Also sections were analyzed for elastin orientation and quantity. Significantly more parallel aligned collagen bundles were found after cyclical (COI = 0.57) and continuous stretch (COI = 0.57) compared with nonstretched skin (COI = 0.40). Similarly, more parallel aligned elastin was found after stretch. Also, significantly thicker collagen bundles and more bundle spacing were found after stretch. For stretched scars, significantly more parallel aligned collagen was found (COI = 0.61) compared with nonstretched scars (COI = 0.49). In conclusion, both elastin and collagen realign in a parallel fashion in response to stretch. For healthy skin, thicker bundles and more space between the bundles were found. Rapid changes in extension, alignment, and collagen morphology appear to be the underlying mechanisms of adaptation to stretching.

Surgery-induced reactive oxygen species enhance colon carcinoma cell binding by disrupting the liver endothelial cell lining.

OBJECTIVE: Resection of primary colorectal cancer is associated with enhanced risk of development of liver metastases. It was previously demonstrated that surgery initiated an early inflammatory response resulting in elevated tumour cell adhesion in the liver. Because reactive oxygen species (ROS) are shown to be produced and released during surgery, the effects of ROS on the liver vascular lining and tumour cell adhesion were investigated. METHODS: Human endothelial cell monolayers (human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells of the lung (HMEC-1s)) were exposed to ROS production, after which electrical impedance, cellular integrity and tumour cell adhesion were investigated. Furthermore, surgery-induced tumour cell adhesion as well as the role of ROS and liver macrophages (Kupffer cells) in this process were studied in vivo. RESULTS: Production of ROS decreased cellular impedance of endothelial monolayers dramatically. Moreover, formation of intercellular gaps in endothelial monolayers was observed, exposing subendothelial extracellular matrix (ECM) on which colon carcinoma cells adhered via integrin molecules. Endothelial damage was, however, prevented in the presence of ROS-scavenging enzymes. Additionally, surgery induced downregulation of both rat and human liver tight junction molecules. Treatment of rats with the ROS scavenger edaravone prevented surgery-induced tumour cell adhesion and downregulation of tight junction proteins in the liver. Interestingly, depletion of Kupffer cells prior to surgery significantly reduced the numbers of adhered tumour cells and prevented disruption of expression of tight junction proteins. CONCLUSIONS: In this study it is shown that surgery-induced ROS production by macrophages damages the vascular lining by downregulating tight junction proteins. This leads to exposure of ECM, to which circulating tumour cells bind. In light of this, perioperative therapeutic intervention, preventing surgery-induced inflammatory reactions, may reduce the risk of developing liver metastases, thereby improving the clinical outcome of patients with colorectal cancer.

Phytosphingosine kills Candida albicans by disrupting its cell membrane.

The mechanism of action of phytosphingosine (PHS), a member of the sphingosine family which has candidacidal activity when added externally, was investigated. Previously, it has been reported that the fungicidal activity of PHS is based on the induction of caspase-independent apoptosis. In contrast, we found that addition of PHS causes a direct permeabilization of the plasma membrane of yeast, highlighted by the influx of the membrane probe propidium iodide, and the efflux of small molecules (i.e., adenine nucleotides) as well as large cellular constituents such as proteins. Freeze-fracture electron microscopy revealed that PHS treatment causes severe damage of the plasma membrane of the cell, which seems to have lost its integrity completely. We also found that PHS reverts the azide-induced insensitivity to histatin 5 (Hst5) of Candida albicans. In a previous study, we had found that the decreased sensitivity to Hst5 of energy-depleted cells is due to rigidification of the plasma membrane, which could be reverted by the membrane fluidizer benzyl alcohol. In line with the increased membrane permeabilization and ultrastructural damage, this reversal of the azide-induced insensitivity by PHS also points to a direct interaction between PHS and the cytoplasmic membrane of C. albicans.

The GTPase activity of Escherichia coli FtsZ determines the magnitude of the FtsZ polymer bundling by ZapA in vitro.

FtsZ polymerizes in a ring-like structure at mid cell to initiate cell division in Escherichia coli. The ring is stabilized by a number of proteins among which the widely conserved ZapA protein. Using antibodies against ZapA, we found surprisingly that the cellular concentration of ZapA is approximately equal to that of FtsZ. This raised the question of how the cell can prevent their interaction and thereby the premature stabilization of FtsZ protofilaments in nondividing cells. Therefore, we studied the FtsZ-ZapA interaction at the physiological pH of 7.5 instead of pH 6.5 (the optimal pH for FtsZ polymerization), under conditions that stimulate protofilament formation (5 mM MgCl(2)) and under conditions that stimulate and stabilize protofilaments (10 mM MgCl(2)). Using pelleting, light scattering, and GTPase assays, it was found that stabilization and bundling of FtsZ polymers by ZapA was inversely correlated to the GTPase activity of FtsZ. As GTP hydrolysis is the rate-limiting factor for depolymerization of FtsZ, we propose that ZapA will only enhance the cooperativity of polymer association during the transition from helical filament to mid cell ring and will not stabilize the short single protofilaments in the cytoplasm. All thus far published in vitro data on the interaction between FtsZ and ZapA have been obtained with His-ZapA. We found that in our case the presence of a His tag fused to ZapA prevented the protein to complement a DeltazapA strain in vivo and that it affected the interaction between FtsZ and ZapA in vitro.

Differences in collagen architecture between keloid, hypertrophic scar, normotrophic scar, and normal skin: An objective histopathological analysis.

Normotrophic, hypertrophic, and keloidal scars are different types of scar formation, which all need a different approach in treatment. Therefore, it is important to differentiate between these types of scar, not only clinically but also histopathologically. Differences were explored for collagen orientation and bundle thickness in 25 normal skin, 57 normotrophic scar, 56 hypertrophic scar, and 56 keloid biopsies, which were selected on clinical diagnosis. Image analysis was performed by fast fourier transformation. The calculated collagen orientation index ranged from 0 (random orientation) to 1 (parallel orientation). The bundle distance was calculated by the average distance between the centers of the collagen bundles. The results showed that compared with all three types of scars, the collagen orientation index was significantly lower in normal skin, which indicates that scars are organized in a more parallel manner. No differences were found between the different scars. Secondly, compared with normal skin, normotrophic scar, and hypertrophic scar, the bundle distance was significantly larger in keloidal scar, which suggests that thicker collagen bundles are present in keloidal scar. This first extensive histological study showed objective differences between normal skin, normotrophic, hypertrophic, and keloidal scar.

Suture-free laser-assisted vessel repair using CO2 laser and liquid albumin solder.

Numerous studies have shown that the use of proteinic solders during laser-assisted vascular anastomosis (LAVA) and repair (LAVR) can significantly increase welding strength, but these studies combined solder-mediated LAVA/R with the use of stay sutures, thereby defeating its purpose. In an in vitro study, we examined the leaking point pressures (LPPs) and histological damage profile of porcine carotid arteries following albumin solder-mediated CO(2) LAVR without the use of sutures. Longitudinal arteriotomies (9.1+/-0.8 mm in length) were sheathed with 25% liquid bovine serum albumin solder, and LAVR was performed using a micromanipulator-controlled CO(2) laser operating at 170-mW power and 1.25-mm spot size in continuous wave mode. The welding regime consisted of a transversal zigzag pass followed by one or two longitudinal zigzag passes, producing an irradiance of 13.9 W/cm(2) and energies of 10.5 J and 11.3 J per mm weld, respectively. LPPs were measured by the fluid infusion technique, and histological analysis was performed with light, fluorescence, and polarization microscopy. The LPP of the two-pass welds was 351+/-158 mmHg versus 538+/-155 mmHg for the three-pass welds. Thermal damage was confined primarily to the adventitial layers, with limited heat diffusion into the media below the solder around the coaptation interface.

Morphology of age-related cuneiform cortical cataracts: the case for mechanical stress.

We evaluated the gross morphology, location, and fiber cell architecture of equatorial cortical opacities in the aging human lens. Using dark-field stereomicroscopy, we photographed donor lenses in toto and as thick slices. In addition, we investigated the details of the fiber cell architecture using fluorescent staining for membranes and by scanning electron microscopy. We then combined our data with data from recent studies on lens viscoelasticity. We found that small cortical and cuneiform opacities are accompanied by changes in fiber structure and architecture mainly in the equatorial border zone between the lens nucleus and cortex. Because the lens cortex and nucleus have different viscoelastic properties in young and old lenses, we hypothesize that external forces during accommodation cause shear stress predominantly in this border zone. The location of the described changes suggests that these mechanical forces may cause fiber disorganization, small cortical opacities, and ultimately, cuneiform cataracts.

Microstructural differences between single-species and dual-species biofilms of Streptococcus mutans and Veillonella parvula, before and after exposure to chlorhexidine.

Dual-species biofilms of Streptococcus mutans and Veillonella parvula are less susceptible to antimicrobials than single-species biofilms of the same microorganisms. The microstructure of single and dual-species biofilms of S. mutans and/or V. parvula was visualized to find out whether the spatial arrangement of bacteria in biofilms is related to survival strategies against antimicrobials. Biofilms were grown in glass-bottomed 96-well microtiter plates and exposed to chlorhexidine at 48 h. Fluorescent probes were used for staining. The microstructure of biofilms was analyzed by confocal scanning laser microscopy at 48, 96, 192, and 264 h. Spatial arrangement analysis was performed using DAIME software for 48 h biofilms. A decrease in the viability and thickness in all types of biofilms was detected after chlorhexidine treatment in time. In untreated biofilms, clustering was observed. In chlorhexidine-treated single-species biofilms, bacteria were dispersed. However, the most prominent clustering was observed in chlorhexidine-treated dual-species biofilm bacteria, which had a higher survival rate compared with chlorhexidine-treated single-species biofilms. Bacteria in dual-species biofilms establish a specific spatial arrangement, forming clusters within distances below 1.2 microm as a survival strategy against antimicrobials while the same bacteria lack this defensive construction in a single-species biofilm.

Spatial segregation of transport and signalling functions between human endothelial caveolae and lipid raft proteomes.

Lipid rafts and caveolae are biochemically similar, specialized domains of the PM (plasma membrane) that cluster specific proteins. However, they are morphologically distinct, implying different, possibly complementary functions. Two-dimensional gel electrophoresis preceding identification of proteins by MS was used to compare the relative abundance of proteins in DRMs (detergent-resistant membranes) isolated from HUVEC (human umbilical-vein endothelial cells), and caveolae immunopurified from DRM fractions. Various signalling and transport proteins were identified and additional cell-surface biotinylation revealed the majority to be exposed, demonstrating their presence at the PM. In resting endothelial cells, the scaffold of immunoisolated caveolae consists of only few resident proteins, related to structure [CAV1 (caveolin-1), vimentin] and transport (V-ATPase), as well as the GPI (glycosylphosphatidylinositol)-linked, surface-exposed protein CD59. Further quantitative characterization by immunoblotting and confocal microscopy of well-known [eNOS (endothelial nitric oxide synthase) and CAV1], less known [SNAP-23 (23 kDa synaptosome-associated protein) and BASP1 (brain acid soluble protein 1)] and novel [C8ORF2 (chromosome 8 open reading frame 2)] proteins showed different subcellular distributions with none of these proteins being exclusive to either caveolae or DRM. However, the DRM-associated fraction of the novel protein C8ORF2 (approximately 5% of total protein) associated with immunoseparated caveolae, in contrast with the raft protein SNAP-23. The segregation of caveolae from lipid rafts was visually confirmed in proliferating cells, where CAV1 was spatially separated from eNOS, SNAP-23 and BASP1. These results provide direct evidence for the previously suggested segregation of transport and signalling functions between specialized domains of the endothelial plasma membrane.

Limited contribution of claudin-5-dependent tight junction strands to endothelial barrier function.

Members of the claudin family are involved in formation of barriers that control access to the paracellular space of epithelia. Likewise, endothelium-specific claudin-5 is involved in the function of the blood-brain barrier (BBB). Here, we assessed the role of claudin-5 in non-BBB endothelial barriers using lentiviral-driven overexpression and silencing of claudin-5 in its native environment of primary vascular endothelial cells. Effects were monitored using macromolecular tracers between 342Da and 40kDa. Measurements were made both in absence and presence of transmigrating leukocytes. Freeze-fracture preparations were analyzed for effects at the ultrastructural level. We show that overexpression of claudin-5 leads to formation of elaborate networks of junction strands, which are absent in untransduced endothelial cells. Concomitantly, a modest, non-size-selective enhancement of the barrier function was observed. In contrast, silencing of endogenous claudin-5 does not influence barrier function. The efficient sealing of the endothelium during diapedesis of monocytes or granulocytes is also claudin-5 independent. Collectively, these data provide evidence for a limited contribution of claudin-5 to the barrier function of human umbilical vein endothelial cells (HUVEC), implying that, unlike selective barriers in epithelia, the barrier of non-BBB endothelium seems largely independent of claudin-directed tight junction structures.

Improved localization of glucose-6-phosphate dehydrogenase activity in cells with 5-cyano-2,3-ditolyl-tetrazolium chloride as fluorescent redox dye reveals its cell cycle-dependent regulation.

Since the introduction of cyano-ditolyl-tetrazolium chloride (CTC), a tetrazolium salt that gives rise to a fluorescent formazan after reduction, it has been applied to quantify activity of dehydrogenases in individual cells using flow cytometry. Confocal laser scanning microscopy (CLSM) showed that the fluorescent formazan was exclusively localized at the surface of individual cells and not at intracellular sites of enzyme activity. In the present study, the technique has been optimized to localize activity of glucose-6-phosphate dehydrogenase (G6PD) intracellularly in individual cells. Activity was demonstrated in cultured fibrosarcoma cells in different stages of the cell cycle. Cells were incubated for the detection of G6PD activity using a medium containing 6% (w/v) polyvinyl alcohol, 5 mM CTC, magnesium chloride, sodium azide, the electron carrier methoxyphenazine methosulphate, NADP, and glucose-6-phosphate. Before incubation, cells were permeabilized with 0.025% glutaraldehyde. Fluorescent formazan was localized exclusively in the cytoplasm of fibrosarcoma cells. The amount of fluorescent formazan in cells increased linearly with incubation time when measured with flow cytometry and CLSM. When combining the Hoechst staining for DNA with the CTC method for the demonstration of G6PD activity, flow cytometry showed that G6PD activity of cells in S phase and G2/M phase is 27 +/- 4% and 43 +/- 4% higher, respectively, than that of cells in G1 phase. CLSM revealed that cells in all phases of mitosis as well as during apoptosis contained considerably lower G6PD activity than cells in interphase. It is concluded that posttranslational regulation of G6PD is responsible for this cell cycle-dependent activity.

Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane.

The effects of antimicrobial peptides on artificial membranes have been well-documented; however, reports on the ultrastructural effects on the membranes of micro-organisms are relatively scarce. We compared the effects of histatin 5 and LL-37, two antimicrobial peptides present in human saliva, on the functional and morphological properties of the Candida albicans cell membrane. Fluorescence microscopy and immunogold transmission electron microscopy revealed that LL-37 remained associated with the cell wall and cell membrane, whereas histatin 5 transmigrated over the membrane and accumulated intracellularly. Freeze-fracture electron microscopy revealed that LL-37 severely affected the membrane morphology, resulting in the disintegration of the membrane bilayer into discrete vesicles, and an instantaneous efflux of small molecules such as ATP as well as larger molecules such as proteins with molecular masses up to 40 kDa. The effects of histatin 5 on the membrane morphology were less pronounced, but still resulted in the efflux of nucleotides. As the morphological defects induced by histatin 5 are much smaller than those induced by LL-37, but the efflux of nucleotides is similar at comparable candidacidal concentrations, we suggest that the loss of nucleotides plays an important role in the killing process.

Different interactions of egg-yolk phosphatidylcholine and sphingomyelin with detergent bile salts.

To examine physical-chemical aspects of bile salt-phospholipid interactions that could contribute to preferential phosphatidylcholine (PC) secretion into bile, we have compared transitions between vesicles and micelles in model systems containing taurocholate (TC) and either egg-yolk PC (EYPC), egg-yolk sphingomyelin (EYSM), buttermilk SM (BMSM) or dipalmitoyl PC (DPPC). Phase transitions from micelles to vesicles were observed at 4-fold dilution of serially diluted EYPC/TC systems, but not earlier than at 16-fold dilution of SM/TC or DPPC/TC systems, indicating lower concentrations of the detergent required for micellization in the case of SM or DPPC. Cryo-transmission electron microscopy of phase transitions initiated by addition of TC to phospholipid vesicles revealed extremely long SM-containing intermediate structures, but shorter EYPC-containing intermediate structures. Again, larger amounts of bile salt were required to induce phase transitions in the case of EYPC compared to SM. Sizes of TC-phospholipid micelles increased progressively upon increasing phospholipid contents in the rank order: DPPC-TC

Ultrastructural effects of antimicrobial peptides from bovine lactoferrin on the membranes of Candida albicans and Escherichia coli.

Antimicrobial peptides allegedly exert their action on microbial membranes. Bovine lactoferrin enfold two antimicrobial domains, lactoferricin B (LFcin B) and lactoferrampin (LFampin). Effects of representative peptides thereof on the membranes of Candida albicans and Escherichia coli were investigated. Confocal laser scanning microscopy revealed that these peptides were internalized within a few minutes, concurrently with disrupting membrane integrity as indicated by freeze-fracture transmission electron microscopy. The most striking findings were induction of distinct vesicle-like structures in the membrane of C. albicans by the LFampin peptide, and detachment of the outer membrane and surface protrusions in E. coli by the LFcin B peptide.

Long-term results of a clinical trial on dermal substitution. A light microscopy and Fourier analysis based evaluation.

Although dermal substitution is a main topic of current wound healing research, there is a paucity of clinical trials with a long-term clinical and histopathological evaluation. A clinical trial was conducted to perform an intra-individual comparison of conventional treatment (split-thickness autograft) to a collagen/elastin dermal substitute in combination with an autograft. Promising results with this substitute were obtained with respect to dermal organisation and scar elasticity in animal studies and clinical trials with a short-term follow-up. Twenty-nine of the 42 pairs of the burn wounds and 28 of the 44 pairs of the scar reconstructions enrolled in the study were biopsied after 1 year. Promising but not statistically significant differences were found between substituted groups and control groups for epidermal thickness, basement membrane maturation, rete ridges (P=0.055), fibroblasts, myofibroblasts, inflammatory cells, vessels and extracellular matrix maturation. An objective and accurate technique, Fourier analysis, was used to evaluate collagen bundle orientation and packing. However, no statistically significant differences were obtained for these parameters. This microscopic evaluation provided no convincing evidence for a long-term effectiveness of a dermal substitute despite promising data over a short-term in in vitro and in vivo studies with the same material.

Collagen morphology in human skin and scar tissue: no adaptations in response to mechanical loading at joints.

Dermal collagen displays a random-like structure that has a major role in strength and function of the human integument. It is hypothesised that collagen bundles align in a parallel fashion in the direction of mechanical tension during scarring, which may explain the problematic scar formation that occurs specifically at joints. Scar tissue and normal skin were biopsied from joints and control areas and evaluated by the Fourier analysis. Collagen orientation was represented by an index ranging from 0 (perfectly random) to 1 (perfectly parallel). Collagen bundle packing signifies the average distance between the centres of collagen bundles. No differences were shown in collagen morphology of scar tissue and normal skin between joints and control areas. Normal skin had a significantly lower collagen orientation index than scar tissue (0.26 versus 0.44, P<0.001). The bundle packing of scar tissue differed significantly from normal skin (18.1 microm versus 23.7 microm, P<0.001). Collagen appeared less parallel orientated in deep dermis compared to superficial dermis especially for normal skin (0.27 versus 0.33, P=0.06). Normal skin had a less parallel organisation in sections that were cut parallel compared to those that were cut perpendicular to the epidermis (0.24 versus 0.30, P=0.02). Collagen orientation of scar tissue is more parallel compared to normal skin. Morphology differs with respect to superficial and deep dermal layers and parallel and perpendicular planes, but appears not to respond to mechanical tension.

Antimicrobial and antibiofilm activity of LL-37 and its truncated variants against Burkholderia pseudomallei.

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 µM LL-31 can reach the bactericidal endpoint within 2h. 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.