Antimicrobial peptides containing unnatural amino acid exhibit potent bactericidal activity against ESKAPE pathogens.

A series of 36 synthetic antimicrobial peptides containing unnatural amino acids were screened to determine their effectiveness to treat Enterococcus faecium, Staphylococcus aureus, Klebsiella pnemoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (ESKAPE) pathogens, which are known to commonly infect chronic wounds. The primary amino acid sequences of these peptides incorporate either three or six dipeptide units consisting of the unnatural amino acids Tetrahydroisoquinolinecarboxylic acid (Tic) and Octahydroindolecarboxylic acid (Oic). The Tic-Oic dipeptide units are separated by SPACER amino acids with specific physicochemical properties that control how these peptides interact with bacterial cell membranes of different chemical compositions. These peptides exhibited minimum inhibitory concentrations (MIC) against these pathogens in the range from >100 to 6.25 µg/mL. The observed diversity of MIC values for these peptides against the various bacterial strains are consistent with our hypothesis that the complementarity of the physicochemical properties of the peptide and the lipid of the bacteria's cell membrane determines the resulting antibacterial activity of the peptide.

Pathogenicity of exopolysaccharide-producing Actinomyces oris isolated from an apical abscess lesion.

AIM: To demonstrate a capacity for producing exopolysaccharides (EPSs) and an ability to form biofilm on abiotic materials of Actinomyces oris strain K20. METHODOLOGY: The productivity of EPSs and the ability to form biofilm of strain K20 were evaluated by measuring viscosity of spent culture media and by scanning electron microscopy (SEM) and the biofilm assay on microtitre plates, respectively. High-performance liquid chromatography was used to determine the chemical composition of the viscous materials. To examine the role of the viscous materials attributable to the pathogenicity in this organism, the ability of strain K20 to induce abscess formation was compared in mice to that of ATCC 27044. RESULTS: The viscosity of the spent culture media of K20 was significantly higher than that of ATCC 27044. Strain K20 showed dense meshwork structures around the cells and formed biofilms on microtitre plates, whereas ATCC 27044 did not. Chemical analysis of the viscous materials revealed that they were mainly composed of neutral sugars with mannose constituting 77.5% of the polysaccharides. Strain K20 induced persistent abscesses in mice lasting at least 5 days at a concentration of 10(8) cells mL(-1), whereas abscesses induced by ATCC 27044 healed and disappeared or decreased in size at day 5. CONCLUSIONS: Strain K20 produced EPSs, mainly consisting of mannose, and formed biofilms. This phenotype might play an important role for A. oris to express virulence through the progression of apical periodontitis.

The effect of lactoferrin on oral bacterial attachment.

INTRODUCTION: Lactoferrin (Lf), an iron-binding salivary glycoprotein, plays an important role in human innate defense against local mucosal infection. We hypothesized that Lf interferes with initial oral bacterial attachment to surfaces by iron sequestration, so inhibiting subsequent biofilm formation. The objective was to investigate the effect of Lf on the early stages of single-species and multi-species oral biofilm development. METHODS: Streptococcus gordonii, Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis were used in this study. Glass disks of a two-track flow cell coated with flowing artificial saliva (0.3 ml/min) with and without Lf (100 microg/ml) were used for studying bacterial attachment (3 h, 37 degrees C). Attachment was also examined by incubating single or multiple species of test bacteria (10(7) colony-forming units/ml) with Lf-coated (20-100 microg/ml) and uncoated glass slides. The effects of beta-lactoglobulin, 2,2'-dipyridyl (25-100 microg/ml), an iron chelator, and FeCl3 on attachment were also examined. RESULTS: Lf inhibited the initial attachment of S. gordonii (50.3%, P < 0.05) but not that of F. nucleatum and P. gingivalis. However, the attachment of a dual-species biofilm containing S. gordonii (i.e. S. gordonii/F. nucleatum or S. gordonii/P. gingivalis) was significantly reduced (48.7% or 62.1%, respectively, P < 0.05) in the presence of Lf. beta-Lactoglobulin did not affect the attachment of S. gordonii. In the presence of 100 microm 2,2'-dipyridyl, attachment of S. gordonii was reduced by 53.87%. No reduction in attachment was noted in S. gordonii pretreated with Lf (100 microg/ml) and FeCl3 (20-200 microm). CONCLUSION: Lf suppresses initial attachment of S. gordonii and S. gordonii coaggregates by iron sequestration. This may lead to subsequent inhibition of oral biofilm development.

The anti-endotoxic effects of the KSL-W decapeptide on Escherichia coli O55:B5 and various oral lipopolysaccharides.

BACKGROUND AND OBJECTIVE: Host responses following the recognition of bacterial lipopolysaccharide can range from acute inflammation to septic shock. The aim of this study was to evaluate the ability of the KSL-W decapeptide to bind to and block the endotoxic effects of lipopolysaccharide. MATERIAL AND METHODS: An enzyme-linked immunosorbent assay-based binding assay using fluorescently labeled KSL-W to detect adsorbed Escherichia coli O55:B5 lipopolysaccharide was employed. A commercially available recombinant Factor C lipopolysaccharide detection assay, hemagglutination of rabbit erythrocytes as well as E-selectin expression in human umbilical vein endothelial cells were used to assess the anti-endotoxic effects after KSL-W exposure to E. coli lipopolysaccharide as well as to oral lipopolysaccharide samples. RESULTS: Lipopolysaccharide-binding assays using E. coli O55:B5 lipopolysaccharide revealed both a higher maximal binding range (532-713 microM) and a half-maximum binding concentration (70-185 microM) for the KSL-W peptide when compared with its analog control. Significant inhibition of E-selectin expression in human umbilical vein endothelial cells (p < 0.0001) as well as hemagglutination of rabbit erythrocytes occurred after the interaction of KSL-W with E. coli lipopolysaccharide. Recombinant Factor C enzyme detection inhibition revealed dose-dependent inhibition values ranging from 1.0-51.8 microM. which were dependent upon the type of lipopolysaccharide sample tested. CONCLUSION: These results demonstrate that for the concentrations tested, the KSL-W decapeptide was nontoxic to mammalian cells and could bind to and block the host recognition and response towards enteric, as well as oral, lipopolysaccharide samples.

Control of oral biofilm formation by an antimicrobial decapeptide.

Oral biofilms are mixed-species microbial communities, and their uncontrolled outgrowth can express as oral diseases. Antimicrobial peptides represent alternative classes of antimicrobials that exhibit selectivity for prokaryotes. We wanted to test the effect of a synthetic decapeptide antimicrobial, KSL, on the development of oral biofilms formed by isolated human salivary bacteria. We used differential interference contrast microscopy, coupled with a dual-flow cell system, to determine the effect of KSL on oral biofilm development. We used reductions of viable counts and confocal microscopy to assess the bactericidal activity of KSL on mature oral biofilms. KSL effectively blocked biofilm development. A significant effect on the viability of mature biofilms was observed when KSL was used in the presence of a surface-active agent, or after biofilms were mechanically disrupted. This study shows that KSL may be a useful adjunct for conventional oral hygiene to prevent plaque-mediated dental diseases.

Phagosomal-phagolysosomal membrane dynamics of stimulated mouse peritoneal macrophages.

Freeze-fracture replication was used to study the membrane events of stimulated mouse peritoneal macrophages during phagocytosis. An increase in intramembrane particles (IMPs) was observed on the protoplasmic fracture (PF) face of the freeze-fractured plasma membrane of phagocytosing macrophages as compared to those of the plasma membrane of nonphagocytosing cells. On the basis of freeze-fracture patterns of membranes, three types of phagosomal membranes were found following the ingestion of the streptomycin-dependent mutant (avirulent) of Salmonella typhimurium (SMD). Most phagosomes 10 min after bacterial uptake had membranes that were structurally similar to the plasma membranes of phagocytosing cells. Structural transformations, i.e., changes in IMP number and size distribution, were observed in phagosomal membranes as the time from bacterial uptake increased. Similar types of phagosomal membranes were also found in phagocytosing macrophages when wild-type Salmonella typhimurium (virulent) was used as the phagocytic challenge. Some indirect morphological evidence suggested that membranes may be pinched off from the phagosomes-phagolysosomes which would be available for recycling back to the cell surface. In the systems studied thus far it appears that the freeze-fracture structure of phagolysosomal membranes is significantly different from that of the plasma membranes. In addition, freeze-fracture evidence suggested that fusion can take place between adjacent phagosomes or phagolysosomes.

Effects of porphyrins and inorganic iron on the growth of Prevotella intermedia.

We demonstrated earlier that hemin-iron-containing compounds which include hemin, human hemoglobin, bovine hemoglobin, and bovine catalase stimulate the growth of Prevotella intermedia [Leung, Subramaniam, Okamoto, Fukushima, Lai, FEMS Microbiol. Lett. 162 (1998) 227-233]. However, the contributions of tetrapyrrole porphyrin ring in these hemin-iron sources as well as inorganic iron for the growth of this organism have not been determined. The purpose of this study was to examine the effects of porphyrins, host iron-binding proteins, and various inorganic iron sources on the growth of hemin-iron depleted P. intermedia. Protoporphyrin IX and protoporphyrin IX-zinc, either in the presence or absence of supplemented ferrous or ferric iron, promoted the growth of P. intermedia at a rate that was comparable to that of the hemin control. On the other hand, neither the host iron proteins, transferrin and lactoferrin, nor the inorganic iron sources which included ferrous chloride, ferric chloride, ferric citrate, ferric nitrate, and ferric ammonium citrate at concentrations up to 200 microM stimulated the growth of hemin-iron-restricted P. intermedia. The results suggest that P. intermedia only use iron in a specific form and that the porphyrin-ring structure is essential for the growth of P. intermedia as in the case of other related organisms.

Hemolytic and hemagglutinating activities of Prevotella intermedia and Prevotella nigrescens.

A total of 91 isolates of Prevotella intermedia or Prevotella nigrescens from subgingival sites were identified by PCR using primers specific for sequences of 16S rRNA. The hemolytic and hemagglutinating activities of the P. intermedia isolates exhibited significantly higher levels compared to those of the P. nigrescens isolates by quantitative analysis. The hemagglutinin gene (phg) was found in 23 of 26 P. intermedia isolates (88.5%), whereas it was found in only two of 44 isolates (4.5%) of P. nigrescens. The high hemolytic and hemagglutinating activities of P. intermedia may be involved in the pathogenicity of P. intermedia in the progression of periodontal disease.

The binding and utilization of hemoglobin by Prevotella intermedia.

Prevotella intermedia, a putative periodontopathic microorganism, requires iron for growth. Hemoglobin can be a major source of iron for bacterial growth in vivo since it is present in the crevicular fluid collected from periodontitis sites. Experiments studying the growth of P. intermedia in iron-depleted Todd-Hewitt broth supplemented with human hemoglobin showed that the bacteria were able to utilize human hemoglobin as a source of iron. The uptake of iron from hemoglobin by P. intermedia appears to be initiated by the binding of hemoglobin to the bacteria as shown by direct binding studies using 125I-labeled human hemoglobin. Scatchard analysis of saturation binding data revealed that 125I-labeled human hemoglobin had a dissociation constant (Kd) of 2.53 x 10(-8) M for the receptor on P. intermedia. Binding of labeled hemoglobin to P. intermedia was competitively inhibited by unlabeled human hemoglobin showing that the binding was specific. The ability of bovine hemoglobin, but not hemin or non-hemoglobin heme-containing compounds, to inhibit binding competitively suggested that the globin moiety of the hemoglobin molecule is recognized by the hemoglobin binding receptors.

Susceptibility of oral bacteria to an antimicrobial decapeptide.

Naturally occurring antimicrobial peptides have emerged as alternative classes of antimicrobials. In general, these antimicrobial peptides exhibit selectivity for prokaryotes and minimize the problems of engendering microbial resistance. As an alternative method to search for more effective broad-spectrum peptide antimicrobials, investigators have developed peptide libraries by using synthetic combinatorial technology. A novel decapeptide, KKVVFKVKFK (KSL), has been identified that shows a broad range of antibacterial activity. The purpose of this study was to test the efficacy of this antimicrobial peptide in killing selected strains of oral pathogens and resident saliva bacteria collected from human subjects. Cytotoxic activity of KSL against mammalian cells and the structural features of this decapeptide were also investigated, the latter by using two-dimensional NMR in aqueous and DMSO solutions. MICs of KSL for the majority of oral bacteria tested in vitro ranged from 3 to 100 microg ml(-1). Minimal bactericidal concentrations of KSL were, in general, within one to two dilutions of the MICs. KSL exhibited an ED(99) (the dose at which 99 % killing was observed after 15 min at 37 degrees C) of 6.25 microg ml(-1) against selected strains of Lactobacillus salivarius, Streptococcus mutans, Streptococcus gordonii and Actinobacillus actinomycetemcomitans. In addition, KSL damaged bacterial cell membranes and caused 1.05 log units reduction of viability counts of saliva bacteria. In vitro toxicity studies showed that KSL, at concentrations up to 1 mg ml(-1), did not induce cell death or compromise the membrane integrity of human gingival fibroblasts. NMR studies suggest that KSL adopts an alpha-helical structure in DMSO solution, which mimics the polar aprotic membrane environment, whereas it remains unstructured in aqueous medium. This study shows that KSL may be a useful antimicrobial agent for inhibiting the growth of oral bacteria that are associated with caries development and early plaque formation.

Localization and identification of root canal bacteria in clinically asymptomatic periapical pathosis.

Twenty-one teeth with clinically asymptomatic periapical pathosis (class 3) were extracted and the isolation, identification, and localization of bacteria in the root apex were examined. Mixtures involving several bacteria were isolated from more than 60% of the cases. Scanning electron microscopy revealed bacterial masses to be associated with the apical part of the root canal, but not with the area of apical foramen or on the surface of root apex. Our results indicate that the bacteria in class 3 cases may be derived from organisms which colonized before or during endodontic treatment, but not from anachoresis. The bacteria-positive cases of asymptomatic periapical pathosis have the potential to progress to symptomatic periapical pathosis.

Intracerebral haemorrhage and Qigong.

We report on a 65-year-old woman who presented with acute right-sided weakness because of an intracerebral (thalamic) haemorrhage. As a Qigong enthusiast with a long-standing history of hypertension, she developed a stroke syndrome soon after practising Qigong one morning. Following neurological recovery, the patient exhibited erratic blood pressure responses while practising Qigong, despite the fact that resting blood pressure was normal. The haemodynamic responses to exercise are discussed and a review of the therapeutic implications of practising Qigong is presented.

Poisoning due to common household products.

From 1988 to 1992, 187 patients were admitted in four general medical wards at the Prince of Wales Hospital, Hong Kong with poisoning due to common household products. The main agents involved included "Dettol" liquid (46%), cleaning products (19%), pesticides (14%), and shampoos (10%). The majority of patients had only relatively mild symptoms. Ingestion of Dettol liquid and strong corrosives tended to be associated with serious complications and deaths. Two patients died after aspiration of Dettol liquid and detergent before and/or during gastric lavage. One other patient died after swallowing sulphuric acid. As with all poisoning, it is very important that the airway is adequately protected before gastric lavage is performed.

Release of a wound-healing agent from PLGA microspheres in a thermosensitive gel.

The purpose of this research was to develop a topical microsphere delivery system in a thermosensitive 20% poloxamer 407 gel (Pluronic F127) to control release of KSL-W, a cationic antimicrobial decapeptide, for a period of 4-7 days for potential application in combat related injuries. KSL-W loaded microsphere formulations were prepared by a solvent extraction-evaporation method (water-oil-water), with poly (D,L-lactic-co-glycolic acid) (PLGA) (50 : 50, low-weight, and hydrophilic end) as the polymeric system. After optimization of the process, three formulations (A, B, and C) were prepared with different organic to water ratio of the primary emulsion while maintaining other components and manufacturing parameters constant. Formulations were characterized for surface morphology, porous nature, drug loading, in vitro drug release, and antimicrobial activity. Microspheres containing 20% peptide with porous surfaces and internal structure were prepared in satisfactory yields and in sizes varying from 25 to 50 µm. Gels of 20% Pluronic F127, which were liquid at or below 24.6°C and formed transparent films at body temperature, were used as carriers for the microspheres. Rheological studies showed a gelation temperature of 24.6°C for the 20% Pluronic F127 gel alone. Gelation temperature and viscosity of formulations A, B, and C as a function of temperature were very close to those of the carrier. A Franz diffusion cell system was used to study the release of peptide from the microspheres suspended in both, phosphate-buffered saline (PBS) and a 20% Pluronic F127 gel. In vitro release of greater than 50% peptide was found in all formulations in both PBS and the gel, and in one formulation there was a release of 75% in both PBS and the gel. Fractions collected from the release process were also tested for bactericidal activity against Staphylococcus epidermidis using the broth microdilution method and found to provide effective antimicrobial activity to warrant consideration and testing in animal wound models for treating combat-related injuries.

Antimicrobial decapeptide KSL-W attenuates Candida albicans virulence by modulating its effects on Toll-like receptor, human ß-defensin, and cytokine expression by engineered human oral mucosa.

We investigated the toxicity of synthetic antimicrobial decapeptide KSL-W on normal human gingival epithelial cell cultures, its effect on Candida albicans adhesion and growth, and the activation of epithelial cell innate immunity. Our results indicate that KSL-W had no toxic effect on cell adhesion or growth, suggesting its safe use with human cells. Pre-treating C. albicans with KSL-W attenuated the yeast's virulence as demonstrated by its reduced adhesion and growth on engineered human oral mucosa epithelium and the subsequent decreased expression of some innate defense molecules by targeted epithelial cells. Indeed, the expression of Toll-like receptors and human ß-defensins was reduced in tissues infected with KSL-W-treated Candida. Proinflammatory cytokine secretion (IL-1ß and IL-6) by the epithelial cells was also regulated by KSL-W in a manner similar to that of antifungal molecule amphotericin B. These findings therefore show that KSL-W is safe for use with human cells and is able to attenuate Candida virulence by modulating its effects on host innate immunity. This study proposes the potential application of KSL-W peptide as an alternative antifungal agent.