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.

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.

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.

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.

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.

Homeostasis in the vertebrate lens: mechanisms of solute exchange.

The eye lens is avascular, deriving nutrients from the aqueous and vitreous humours. It is, however, unclear which mechanisms mediate the transfer of solutes between these humours and the lens' fibre cells (FCs). In this review, we integrate the published data with the previously unpublished ultrastructural, dye loading and magnetic resonance imaging results. The picture emerging is that solute transfer between the humours and the fibre mass is determined by four processes: (i) paracellular transport of ions, water and small molecules along the intercellular spaces between epithelial and FCs, driven by Na(+)-leak conductance; (ii) membrane transport of such solutes from the intercellular spaces into the fibre cytoplasm by specific carriers and transporters; (iii) gap-junctional coupling mediating solute flux between superficial and deeper fibres, Na(+)/K(+)-ATPase-driven efflux of waste products in the equator, and electrical coupling of fibres; and (iv) transcellular transfer via caveoli and coated vesicles for the uptake of macromolecules and cholesterol. There is evidence that the Na(+)-driven influx of solutes occurs via paracellular and membrane transport and the Na(+)/K(+)-ATPase-driven efflux of waste products via gap junctions. This micro-circulation is likely restricted to the superficial cortex and nearly absent beyond the zone of organelle loss, forming a solute exchange barrier in the lens.

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.

Antimicrobial activities of LL-37 and its truncated variants against Burkholderia thailandensis.

Antimicrobial peptides (AMPs) are essential host defence molecules found in a wide variety of species and are promising antibacterial therapeutic candidates. Focusing on the human cathelicidin peptide LL-37, the aim of the present study was to explore the mechanisms of action and antimicrobial activities of a library of LL-37 fragments using Burkholderia thailandensis E264 as a model. The results revealed that IG-19 was the shortest fragment within LL-37 that exhibited antibacterial activity. LL-31, missing six residues at the C-terminus of LL-37, exhibited the strongest killing effect. Freeze-fracture electron microscopy of bacterial cells treated with either LL-37 or LL-31 revealed irregular bacterial surfaces with bleb projections, indicating that these peptides disrupted the integrity of the membrane. In addition, these peptides induced leakage of cell components, including nucleotides and even proteins. Altogether, the results obtained indicate the potential of using LL-31 as a new AMP to combat Burkholderia spp.

Inside-out regulation of ICAM-1 dynamics in TNF-alpha-activated endothelium.

BACKGROUND: During transendothelial migration, leukocytes use adhesion molecules, such as ICAM-1, to adhere to the endothelium. ICAM-1 is a dynamic molecule that is localized in the apical membrane of the endothelium and clusters upon binding to leukocytes. However, not much is known about the regulation of ICAM-1 clustering and whether membrane dynamics are linked to the ability of ICAM-1 to cluster and bind leukocyte integrins. Therefore, we studied the dynamics of endothelial ICAM-1 under non-clustered and clustered conditions. PRINCIPAL FINDINGS: Detailed scanning electron and fluorescent microscopy showed that the apical surface of endothelial cells constitutively forms small filopodia-like protrusions that are positive for ICAM-1 and freely move within the lateral plane of the membrane. Clustering of ICAM-1, using anti-ICAM-1 antibody-coated beads, efficiently and rapidly recruits ICAM-1. Using fluorescence recovery after photo-bleaching (FRAP), we found that clustering increased the immobile fraction of ICAM-1, compared to non-clustered ICAM-1. This shift required the intracellular portion of ICAM-1. Moreover, biochemical assays showed that ICAM-1 clustering recruited beta-actin and filamin. Cytochalasin B, which interferes with actin polymerization, delayed the clustering of ICAM-1. In addition, we could show that cytochalasin B decreased the immobile fraction of clustered ICAM-1-GFP, but had no effect on non-clustered ICAM-1. Also, the motor protein myosin-II is recruited to ICAM-1 adhesion sites and its inhibition increased the immobile fraction of both non-clustered and clustered ICAM-1. Finally, blocking Rac1 activation, the formation of lipid rafts, myosin-II activity or actin polymerization, but not Src, reduced the adhesive function of ICAM-1, tested under physiological flow conditions. CONCLUSIONS: Together, these findings indicate that ICAM-1 clustering is regulated in an inside-out fashion through the actin cytoskeleton. Overall, these data indicate that signaling events within the endothelium are required for efficient ICAM-1-mediated leukocyte adhesion.

Optical phantoms of varying geometry based on thin building blocks with controlled optical properties.

Current innovations in optical imaging, measurement techniques, and data analysis algorithms express the need for reliable testing and comparison methods. We present the design and characterization of silicone elastomer-based optical phantoms. Absorption is included by adding a green dye and scattering by adding TiO(2) or SiO(2) particles. Optical coherence tomography measurements demonstrate a linear dependence of the attenuation coefficient with scatterer concentration in the absence of absorbers. Optical transmission spectroscopy of the nonscattering absorbing phantoms shows a linear concentration dependent absorption coefficient. Both types of samples are stable over a period of 6 months. Confocal microscopy of the samples demonstrates a homogeneous distribution of the scatterers, albeit with some clustering. Based on layers with thicknesses as small as 50 mum, we make multifaceted structures resembling flow channels, (wavy) skin-like structures, and a layered and curved phantom resembling the human retina. Finally, we demonstrate the ability to incorporate gold nanoparticles within the phantoms. In conclusion, our phantoms are easy to make, are based on affordable materials, exhibit well-defined and controllable thickness, refractive index, absorption, and scattering coefficients, are homogeneous, and allow the incorporation of novel types of nanoparticle contrast agents. We believe our phantoms fulfill many of the requirements for an "ideal" tissue phantom, and will be particularly suited for novel optical coherence tomography applications.

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.

Renal cell carcinoma and oxidative stress: The lack of peroxisomes.

Oxidative stress plays an important role in carcinogenesis because of induction of DNA damage and its effects on intracellular signal transduction pathways. Here, we investigated the relationship between the defence against oxidative stress and human renal cell carcinoma that originates from proximal tubular epithelium. Oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase (G6PD) activity is a diagnostic tool for the detection of carcinomas. Its mechanism is based on high G6PD activity, reduced superoxide dismutase activity and reduced numbers of peroxisomes in the cancer cells. Five out of the 8 renal carcinomas studied here demonstrated oxygen insensitivity. These carcinomas showed high G6PD activity, whereas the other 3 carcinomas contained lower G6PD activity and were oxygen sensitive like non-cancer cells. Oxygen insensitivity did not correlate with tumour grade, staging or presence of metastases. Electron microscopy and immunofluorescence of catalase showed large numbers of peroxisomes in epithelial cells of proximal tubules of normal human kidney, whereas these organelles were completely absent in cancer cells of all carcinomas. As a consequence of the absence of peroxisomes in cancer cells, fatty acid metabolism is disturbed in addition to the altered glucose metabolism that is generally observed in cancer cells. Therefore, therapeutic approaches should focus on metabolism in addition to other strategies targeting signal transduction and angiogenesis.

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.

Bone morphogenetic protein 4 expressed in esophagitis induces a columnar phenotype in esophageal squamous cells.

BACKGROUND & AIMS: Barrett's esophagus (BE) is a metaplastic condition in which normal squamous esophageal epithelium is replaced by columnar epithelium. It is proposed that one of the possible mechanisms is dedifferentiation of squamous epithelium into columnar epithelium. The pathophysiology through which this metaplasia occurs is unknown. A recent study by serial analysis of gene expression showed that bone morphogenetic protein 4 (BMP-4) is uniquely expressed in BE. In this study, the role of the BMP pathway in the metaplastic transformation of normal squamous cells into columnar cells was examined. METHODS: Tissues from patients with esophagitis and BE and in an esophagitis-BE rat model were examined for the activation of the BMP pathway. Short-term cultures of primary normal squamous esophageal cells were treated with BMP-4, and cell biological changes were examined by Western blot analysis, immunohistochemistry, and microarrays. RESULTS: In both human and rat tissues, the BMP pathway proved to be activated in esophagitis and BE. Upon incubation of squamous cell cultures with BMP-4, the cytokeratin expression pattern showed a shift that was consistent with columnar epithelium. Involvement of the BMP pathway was suggested by up-regulation of Phosphorylated-Smad 1/5/8 (P-Smad 1/5/8) that was effectively blocked by Noggin, a BMP antagonist. Comparison of the gene expression profiles of squamous cells, BMP-4-treated squamous cells, and BE cells showed a significant shift in the profile of the BMP-4-treated squamous cells toward that of the cultured BE cells. CONCLUSIONS: These results suggest that the BMP pathway could play a role in the transformation of normal esophageal squamous cells into columnar cells.

Development and adult morphology of the eye lens in the zebrafish.

The zebrafish has become an important vertebrate model organism to study the development of the visual system. Mutagenesis projects have resulted in the identification of hundreds of eye mutants. Analysis of the phenotypes of these mutants relies on in depth knowledge of the embryogenesis in wild-type animals. While the morphological events leading to the formation of the retina and its connections to the central nervous system have been described in great detail, the characterization of the development of the eye lens is still incomplete. In the present study, we provide a morphological description of embryonic and larval lens development as well as adult lens morphology in the zebrafish. Our analyses show that, in contrast to other vertebrate species, the zebrafish lens delaminates from the surface ectoderm as a solid cluster of cells. Detachment of the prospective lens from the surface ectoderm is facilitated by apoptosis. Primary fibre cell elongation occurs in a circular fashion resulting in an embryonic lens nucleus with concentric shells of fibres. After formation of a monolayer of lens epithelial cells, differentiation and elongation of secondary lens fibres result in a final lens morphology similar to that of other vertebrate species. As in other vertebrates, secondary fibre cell differentiation includes the programmed degradation of nuclei, the interconnection of adjacent fibres via protrusions at the fibre cells' edges and the establishment of gap junctions between lens fibre cells. The very close spacing of the nuclei of the differentiating secondary fibres in a narrow zone close to the equatorial epithelium, however, suggests that secondary fibre cell differentiation deviates from that described for mammalian or avian lenses. In summary, while there are similarities in the development and final morphology of the zebrafish lens with mammalian and avian lenses, there are also significant differences, suggesting caution when extrapolating findings on the zebrafish to, for example, human lens development or function.

Energy depletion protects Candida albicans against antimicrobial peptides by rigidifying its cell membrane.

Inhibitors of the energy metabolism, such as sodium azide and valinomycin, render yeast cells completely resistant against the killing action of a number of cationic antimicrobial peptides, including the salivary antimicrobial peptide Histatin 5. In this study the Histatin 5-mediated killing of the opportunistic yeast Candida albicans was used as a model system to comprehensively investigate the molecular basis underlying this phenomenon. Using confocal and electron microscopy it was demonstrated that the energy poison azide reversibly blocked the entry of Histatin 5 at the level of the yeast cell wall. Azide treatment hardly induced depolarization of the yeast cell membrane potential, excluding it as a cause of the lowered sensitivity. In contrast, the diminished sensitivity to Histatin 5 of energy-depleted C. albicans was restored by increasing the fluidity of the membrane using the membrane fluidizer benzyl alcohol. Furthermore, rigidification of the membrane by incubation at low temperature or in the presence of the membrane rigidifier Me(2)SO increased the resistance against Histatin 5, while not affecting the energy charge of the cell. In line, azide induced alterations in the physical state of the interior of the lipid bilayer. These data demonstrate that changes in the physical state of the membrane underlie the increased resistance to antimicrobial peptides.

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.