Nanoparticle-Encapsulated Chlorhexidine against Oral Bacterial Biofilms

Background

Chlorhexidine (CHX) is a widely used antimicrobial agent in dentistry. Herein, we report the synthesis of a novel mesoporous silica nanoparticle-encapsulated pure CHX (Nano-CHX), and its mechanical profile and antimicrobial properties against oral biofilms.

Methodology/Principal Findings

The release of CHX from the Nano-CHX was characterized by UV/visible absorption spectroscopy. The antimicrobial properties of Nano-CHX were evaluated in both planktonic and biofilm modes of representative oral pathogenic bacteria. The Nano-CHX demonstrated potent antibacterial effects on planktonic bacteria and mono-species biofilms at the concentrations of 50–200 µg/mL against Streptococcus mutans, Streptococcus sobrinus, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Enterococccus faecalis. Moreover, Nano-CHX effectively suppressed multi-species biofilms such as S. mutans, F. nucleatum, A. actinomycetemcomitans and Porphyromonas gingivalis up to 72 h.

Conclusions/Significance

This pioneering study demonstrates the potent antibacterial effects of the Nano-CHX on oral biofilms, and it may be developed as a novel and promising anti-biofilm agent for clinical use.     

Pseudomonas putida strain PCL1760 controls tomato foot and root rot in stonewool under industrial conditions in a certified greenhouse

Pseudomonas putida strain PCL1760 was isolated previously from the avocado rhizosphere, using an enrichment method for competitive tomato root tip colonizers that selects for biological control strains which act through the biological control mechanism “competition for nutrients and niches” (CNN). Here we demonstrate that strain PCL1760 showed significant biological control of tomato foot and root rot (TFRR), a disease caused by Fusarium oxysporum f. sp. radicis-lycopersici (Forl), in eight independent laboratory experiments in stonewool substrate. Furthermore, its activity in stonewool was also tested in an industrial setup. The presence of Forl appeared to decrease seed germination. The additional presence of the biological control strain partly restored the germination level. Introduction of P. putida PCL1760 resulted in significant biological control of TFRR. PCR quantification revealed that the biological control strain decreased the amount of Forl DNA in tomato plant tissue significantly. We conclude that the results of this trial show that P. putida strain PCL1760, which acts through the new mechanism CNN, controls TFRR also under industrial stonewool conditions.     

Synergistic activity of chlorhexidine and synoeca-MP peptide against Pseudomonas aeruginosa

‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬This study aims to evaluate the in vitro antimicrobial and immunomodulatory activities and cytotoxicity of chlorhexidine (CHX) and synoeca-MP peptide alone or in combination against Pseudomonas aeruginosa. The antimicrobial property was evaluated by the determination of minimal inhibitory concentration, minimum bactericidal concentration, and planktonic bacteria and biofilm inhibition. Immunomodulatory activity was determined by enzyme-linked immunosorbent assay and nitric oxide production by the Griess reaction method. According to the results, synoeca-MP combined with CHX demonstrated antimicrobial effectiveness compared with its isolated use, in addition to immunomodulatory activity (upregulating MPC-1 and tumor necrosis factor-α and downregulating nitric oxide and interleukin-10). In this context, it is expected that the substances, together, could be capable of controlling bacterial infection and dissemination, besides potentiating macrophages’ immune response against the studied microorganism. Moreover, reducing the CHX concentration by the addition of synoeca-MP peptide may, in a beneficial way, minimize the undesirable effects of both, CHX and synoeca-MP in a clinical setting.     

Studies on base-exchange reactions of phospholipids in rat brain particles and a "solubilized" system.

A filtration-method on Millipore-membranes for the assay of the base-exchange reaction was described. Its advantage over the usual procedure based upon the extraction and the washing of lipids was discussed with the viewpoint of processing many samples, which would be indispensable for purifying the enzyme.The reaction showed an absolute dependency for calcium ion with different optimal concentrations for each of the three bases, a sensitivity to inhibition by high ionic strength, and a pH optimum around 9.0. Exogenously added phospholipid, asolectin, gave a slight stimulation for ethanolamine and l-serine incorporation at a low concentration while choline incorporation was essentially inhibited at all concentrations examined. In heat-denaturation experiments with the particulate and soluble the incorporation of choline into lipid was more sensitive than that of ethanolamine and l-serine. A developmental study showed that brain particles sedimenting between 10,000 and 35,000g prepared from rats aged 22–27 days readily incorporated ethanolamine, l-serine, and choline into their corresponding phosphatidyl compound.Several procedures for solubilization of the “base-exchange” enzyme were examined. The most effectively solubilized preparation was obtained by the use of an ionically balanced detergent, Miranol H2M. This preparation showed a marked dependency on exogenously added phospholipids for its maximal enzymic activity, had a pH optimum at around 7.2, and had an absolute requirement for Ca²⁺. This particular detergent at a concentration of 1% ($\text{w}\text{v}$) solubilized approximately 50% of the protein, and about 30% of the phospholipids, 40% of the cerebrosides, and only 11% of the cholesterol originally present in the particles. The relative proportions of different phospholipids solubilized by the detergent were, however, similar to those present in the original particles.The base-exchange reaction catalyzed by the solubilized enzyme was found to be highly sensitive to ionic strength, and the inhibitory effect of a specific monovalent cation paralleled its ionic size. Substantial differences in the K_m value for each of the substrates with only slight differences in V were observed.The choice of solubilizing agents in relation to these properties and to the maintenance of the activity of the base-exchange reaction was discussed.     

Elaboration of immune stimulating lipid-saponin subunit antigen carrier based on glycolipid monogalactosyldiacylglycerol from sea macrophytes and triterpene glycosides from Cucumaria japonica

The ability of some triterpene glycosides of holothurians: holotoxin A₁ from Apostichopus japonicus and a mixture of monosulfated triterpene glycosides from Cucumaria japonica called cucumarioside (CD) to form supramolecular complexes with cholesterol (Chol) and monogalactosyldiacylglycerol (MGDG) or phosphatidylcholine (PC) was studied. A transmission electron microscopy method was used to observe supramolecular lipid-saponin complexes formed by holotoxin A1 and CD with cholesterol in the presence of membrane lipids. The observed supramolecular complexes are tubular nanoparticles with a length of 100–300 nm, an external diameter of 10–16 nm and an internal diameter of 2–6 nm. The formation of tubular nanoparticles was more effective in the presence of MGDG than with PC. Nanoparticles forming in the presence of MGDG are shaped as a tubule, have a constant diameter and a strongly pronounced internal channel. In contrast, PC has no such properties; this lipid is unable to fully integrate in tubular nanoparticles. Based on electron-microscopy data the range of weight ratio of MGDG-Chol-CD was determined as a 1–10: 2: 3 that provided most effective formation of tubular nanoparticles. Different methods of incorporation of model antigens in complex MGDG-Chol-CD were studied. Influenza hemagglutinin and neuraminidase from commercial vaccine “Influvac” and pore forming protein YompF from Yersinia pseudotuberculosis were used as model antigens. From 54 to 72% of protein of “Influvac” vaccine and 88–92% of YompF were incorporated in supramolecular complexes depending on the method of incorporation. The loss of functional activity of hemagglutinin of vaccine “Influvac” was the result of applying ultrasonic disintegration for incorporation of this protein in complex MGDG-Chol-CD. YompF incorporation in MGDG-Chol-CD complex led to the increased diameter of tubular particles, in the same time incorporation of vaccine “Influvac” antigens produced the “cap” formation at the end of tubules. The possibility of a described supramolecular complex MGDG-Chol-CD to be a carrier for subunit bacterial and viral antigens is shown.     

Chemical synthesis and biological evaluation of an antimicrobial peptide gonococcal growth inhibitor

Gonococcal growth inhibitor 1 (GGI-1) is a 44-residue peptide with potent anti-Legionella activity. It has been isolated from Staphylococcus haemolyticus but, to date, its chemical synthesis has not been reported. Acquisition of this peptide via this means would enable a more detailed examination of its antimicrobial properties. However, its synthesis represents a significant challenge because of two predicted “difficult sequences” within the peptide. Its successful solid-phase assembly is reported in this paper, and was accomplished by use of simple palliative measures including the introduction of a single pseudo-proline isostere in order to counteract on-resin aggregation. The peptide had moderate antimicrobial activity against Escherichia coli but was inactive against another Gram-negative bacterium and two Gram-positive bacteria (Bacillus species). It had significant haemolytic activity, with a H₅₀ (concentration of peptide that causes 50?% haemolysis) of 20 and 125?μM for two blood samples from different donors. An alternative therapeutic index to that proposed for GGI-1 in a recent publication is proposed.     

Synthesis of poly(ε-caprolactone) by an immobilized lipase coated with ionic liquids in a solvent-free condition

Polycaprolactone (PCL) was synthesized by ring-opening polymerization of ε-caprolactone through two different enzymatic processes. The lipase from Candida antarctica B, immobilized on macroporous acrylic acid beads, was employed either untreated or coated with small amounts of ionic liquids (ILs). Monocationic ionic liquids, [C _n MIm][NTf₂] (n = 2, 6, 12), as well as a dicationic ionic liquid, ([C₄(C₆Im)₂][NTf₂]₂), were used to coat the immobilized lipase and also as the reaction medium. In both methods, the polarity, anion of the ILs concentration and viscosity strongly influenced the reaction. Coating the immobilized enzyme with ILs improved catalytic activity and less ILs was required to produce PCL with a higher molecular weight and reaction yield. At 60 °C and ILs/Novozyme-435 coating ratio of 3:1 (w/w) for 48 h, the highest M _w and reaction yield of PCL were 35,600 g/mol and 62 % in the case of [C₁₂MIm][NTf₂], while the M _w and reaction yield of PCL was 20,300 g/mol and 54 % with [C₁₂MIm][NTf₂] and catalyzed by untreated lipase.     

Hirticrusta gen. nov. segregated from Neofomitella in Polyporaceae (Polyporales)

Taxonomic studies including morphological observations and phylogenetic analyses were conducted on Japanese “uragin-take”, an unidentified species from Amazonia, Brazil and their allies. Phylogenetic analyses using ITS, nrLSU and RPB2 regions revealed that “uragin-take”, Neofomitella polyzonata and the unidentified species formed a monophyletic clade separate from the clade including the other four Neofomitella spp. and that “uragin-take” is conspecific with N. polyzonata. Morphological investigations on authentic specimens revealed that Polyporus subradiatus is a prior name for N. polyzonata. We propose Hirticrusta gen. nov. typified by H. subradiata segregated from Neofomitella, and we erected H. amazonica sp. nov. for the unidentified species. Hirticrusta is characterized by annual to biennial and sessile basidiocarps, semicircular to dimidiate pileus, velutinous to tomentose hairs on pileus surface, buff to brown context with a crustose layer indicated by a dark brown line forming a longitudinal section below the superficial hairs, a trimitic hyphal system, crustose layer composed of parallel and densely arranged brown hyphae and cylindrical basidiospores. The new species, H. amazonica is distinguishable from other polypores by downy and long tomentum on the pileus surface (up to 20 mm thick), brown context with a dark brown layer below the tomentum and round pores (5–7/mm).     

Killing activity of LFchimera on periodontopathic bacteria and multispecies oral biofilm formation in vitro

Lactoferrin chimera (LFchimera), a heterodimeric peptide containing lactoferrampin (LFampin265–284) and a part of lactoferricin (LFcin17–30), possesses a broad spectrum of antimicrobial activity. However, there is no report on the inhibitory effects of LFchimera against multispecies oral biofilms. This study aimed to determine the effects of LFchimera in comparison to chlorhexidine digluconate (CHX) and minocycline hydrochloride (MH), on in vitro multispecies biofilms derived from subgingival plaque of periodontitis patients harboring Aggregatibacter actinomycetemcomitans. First the effects of LFchimera against planktonic and an 1-day old biofilm of the periodontopathic bacteria, A. actinomycetemcomitans ATCC 43718 were established. Then, the effects on biofilm formation and bacterial viability in the multispecies biofilm were determined by crystal violet staining and LIVE/DEAD BacLight Bacterial Viability kit, respectively. The results revealed that a significant reduction (P?<?0.05) in biofilm formation occurred after 15 min exposure to 20 µM of LFchimera or CHX compared to control. In contrast, MH at concentration up to 100 µM did not inhibit biofilm formation. The ratio of live/dead bacteria in biofilm was also significantly lower after 15 min exposure to 20 µM of LFchimera compared to control and 20–50 µM of CHX and MH. Altogether, the results obtained indicate that LFchimera is able to inhibit in vitro subgingival biofilm formation and reduce viability of multispecies bacteria in biofilm better than CHX and MH.     

Enzymatic grafting of natural phenols to flax fibres: Development of antimicrobial properties

Unbleached flax fibres for paper production were treated with laccase from Pycnoporus cinnabarinus and low molecular weight phenols (syringaldehyde - SA, acetosyringone - AS and p-coumaric acid - PCA) to evaluate the potential of this treatment to biomodify high cellulose content fibres. After the enzymatic treatment with the phenols, an increase in kappa number was found, probably due to a covalent binding of the phenoxy radicals on fibres. Grafting was more evident in pulps treated with PCA (an increase of 4 kappa number points with respect to the laccase control was achieved). Paper handsheets from treated pulps showed antimicrobial activity against the bacteria tested: Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae. An important reduction on microbial count was obtained after incubation of liquid cultures of the bacteria with grafted handsheets. AS and PCA grafted fibres showed a high antibacterial activity on K. pneumoniae, getting a nearly total growth inhibition. AS fibres also caused a high reduction in bacterial population of P. aeruginosa (97% reduction). Optical properties of handsheets from treated pulps were also determined, showing a brightness decrease and increase in coloration, evaluated by CIE L*a*b* system, caused by the laccase induced grafting of the phenols. The results suggest that these low molecular weight phenols, covalently bound to the flax fibres by the laccase treatment, can act as antimicrobial agents and produce handsheets with antimicrobial activity.     

Evaluation of biocompatibility and mechanical behavior of chitin-based polyurethane elastomers. Part-II: Effect of diisocyanate structure

Chitin-based polyurethane elastomers having potential for biomedical applications with tunable mechanical properties were synthesized by step growth polymerization techniques using poly(?-caprolactone) (PCL) with different diisocyanates. The prepolymer was extended using chitin and/or 1,4-butane diol (BDO). The structures of the resulted polymers were determined by Fourier transform infrared (FTIR), ¹H NMR and ¹³C NMR spectroscopic techniques. The effect of structure of diisocyanates and chain extenders on mechanical properties and in vitro biocompatibility were investigated. The results revealed that the final polymers extended with chitin are preferred candidates for surgical threads with on going investigations into their in vitro biocompatibility and non-toxicity.     

Effect of molecular weight of chitosan on antimicrobial properties and tissue compatibility of chitosan-impregnated bacterial cellulose films

Bacterial cellulose-chitosan (BC-C) films were developed by immersing purified BC pellicles in 1.5 ~ 2.0% (w/v) acetic acid solutions containing chitosan of varying molecular weights. Effects of different molecular weight of chitosan on physical, biological and antimicrobial properties of the composite films were investigated. The cumulative chitosan absorption capacities with M_w of 141,000, 199,000, and 263,000 were 38.43, 24.65, and 23.89 mg/cm³ of dry BC film, respectively. The cumulative release profiles of chitosan from the films strongly depended on molecular weight of chitosan and pH of solution. The order of release of chitosan from the BC-C films was dependent on molecular weight as follows: M_w 141,000 > M_w 199,000 > M_w 263,000. All BC-C films showed the antimicrobial abilities against Staphylococcus aureus and Aspergillus niger but had no inhibitory effect on the growth of Escherichia coli. The BC-C films supported for adhesion, spreading and proliferation of both human skin keratinocytes and fibroblasts. The antibacterial activity against S. aureus of the BC-C with the highest Mw chitosan (263,000) was higher than those of the others. On the other hand, the BC-C films with the lowest M_w chitosan (141,000) promoted the growth of human skin cells more than those of the others.     

Different patterns of apoptosis of HL-60 cells induced by cycloheximide and camptothecin

Cells of the human promyelocytic HL-60 line, when treated with a variety of antitumor agents in the presence of the protein synthesis inhibitor cycloheximide (CHX), or with CHX alone, rapidly undergo apoptosis (“active cell death”). It is presumed, therefore, that such cells are “primed” to apoptosis in that no new protein synthesis is required for induction of their death. We have studied apoptosis of HL-60 cells triggered by the DNA topoisomerase I inhibitor camptothecin (CAM) in the absence and presence of CHX and apoptosis induced by CHX alone. Two different flcw cytometric methods were used, each allowing us to relate the apoptosis-associated DNA degradation to the cell cycle position. Apoptosis induced by CAM was limited to S phase cells, e.g., at a CAM concentration of 0.15 μM, nearly 90% of the S phase cells underwent apoptosis after 4 h. In contrast, apoptosis triggered by CHX was indiscriminate, affecting all phases of the cycle: ～40% of the cells from each phase the cycle underwent apoptosis at 5 μM CHX concentration. When CAM and CHX were added together, the pattern of apoptosis resembled that of cycloheximide alone, namely, cells in all phases of the cycle in similar proportion were affected. Thus, CHX, while itself inducing apoptosis of a fraction of cells, protected the S phase cells against apoptosis triggered by CAM. Because CHX (5 μM) did not significantly affect the rate of cell progression through S phase, the observed protective effect was most likely directly related to inhibition of protein synthesis, rather than to its possible indirect effect on DNA replication. Furthermore, whereas apoptosis (DNA degradation) triggered by CAM was prevented by the serine protease inhibitor N-tosyl-L-lysylchloromethyl ketone (TLCK), this process was actually potentiated by this inhibitor when induced by CHX. The present data indicate differences in mechanism of apoptosis triggered by CAM (and perhaps other antitumor drugs) as compared with CHX. Apoptosis caused by CHX may be unique in that it may not involve new protein synthesis. These data are compatible with the assumption that the loss of a hypothetical, rapidly turning over suppressor of apoptosis may be the trigger of apoptosis of HL-60 cells treated with CHX, whereas de novo protein synthesis is required when apoptosis is triggered by other agents. © 1993 Wiley-Liss, Inc.     

Coupling of homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase

Several types of conditions allow the disconnection of homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase. A model that includes a concerted gross conformational change corresponding to the homotropic cooperative interactions between the catalytic sites and local “site by site” effects promoted by the effectors accounts for this disconnection as well as for the other known properties of the enzyme. However, the substrate concentration influences the extent of stimulation and feedback inhibition of the catalytic activity by the effectors. This result is explained by assuming that these effectors promote a “primary effect”, which is exerted locally “site by site”, and a “secondary effect”, which is mediated by the substrate. As predicted by the model, relaxed (R) forms of the enzyme show only the primary effect. In addition 2-ThioU-aspartate transcarbamylase, a modified form of the enzyme in which the homotropic cooperative interactions between the catalytic sites are selectively abolished, shows the same heterogeneity in CTP binding sites as normal aspartate transcarbamylase.     

Antimicrobial activity of disinfectant agents incorporated into type IV dental stone

doi: 10.1111/j.1741‐2358.2011.00462.x
Antimicrobial activity of disinfectant agents incorporated into type IV dental stone Purpose: This study evaluated the antimicrobial activity of two disinfectant agents, 2% chlorhexidine digluconate solution (CHX) and 98% chlorhexidine hydrochloride powder (HYD), incorporated into type IV dental stone at the time of mixing. Material and methods: Agar diffusion test was used for the following microorganisms: Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Candida albicans. The specimens were grouped in: (1) dental stone mixed with sterile distilled water; (2) paper disc soaked with CHX; (3) dental stone mixed with CHX; and (4) dental stone with incorporation of HYD, in 1% proportion of the dental stone mass and mixed with sterile distilled water. The culture medium was inoculated with microbial suspensions 1 and 24 h after pouring of the dental stone. The antimicrobial activity was evaluated by the average diameter of microbial growth inhibition zones. The data were analysed with a nested anova (p < 0.05) and Tukey test for specific comparisons. Results: The disinfectant agents demonstrated antimicrobial activity against all microorganisms, with the exception of C. albicans, against which the CHX was ineffective in two periods of analysis. Significant differences between disinfectants were found with all microorganisms. Conclusion: The disinfectant agents analysed were effective against most of the microorganisms tested, except C. albicans.     

Actividad de la micafungina contra las biopelículas de Candida

BackgroundMost recalcitrant infections are associated to colonization and microbial biofilm development. These biofilms are difficult to eliminate by the immune response mechanisms and the current antimicrobial therapy.AimTo describe the antifungal of micafungin against fungal biofilms based in the scientific and medical literature of recent years.MethodsWe have done a bibliographic retrieval using the scientific terms “micafungin”, “activity”, “biofilm”, “Candida”, “Aspergillus”, “fungi”, “mycos”*, susceptibility, in PubMed/Medline from the National Library of Medicine from 2006 to 2009.ResultsMost current antifungal agents (amphotericin B and fluconazole) and the new azole antifungals have no activity against fungal biofilms. However, micafungin and the rest of echinocandins are very active against Candida albicans, Candida dubliniensis, Candida glabrata, and Candida krusei biofilms but their activities are variable and less strong against Candida tropicalis and Candida parapsilosis biofilms. Moreover, they have not activities against the biofilms of Cryptococcus y Trichosporon.ConclusionsThe activity of micafungin against Candida biofilms gives more strength to its therapeutic indication for candidaemia and invasive candidiasis associated to catheter, prosthesis and other biomedical devices.     

Muscle growth and protein synthesis in the puparia of Calliphora vomitoria

The effects of cycloheximide on the development of the dorsal longitudinal flight muscle of 3- to 5-day-old puparia of Calliphora vomitoria have been investigated. One μg of cycloheximide injected into the puparia reduced the incorporation of ¹⁴C phenylalanine and lysine into protein to 5 and 8 per cent of their normal levels. The cycloheximide was found to have produced its maximum effect within 2 hr of its injection and increasing the concentration did not further depress the amount of amino acid incorporation. The sixth dorsal longitudinal muscles continued to increase in length after the injection of cycloheximide and the elongation of the muscle fibres was accompanied by an increase in protein content in normal and cycloheximide-treated animals. An injection of colchicine (which is believed to disrupt microtubules) immediately halted muscle growth. Electron microscopy of the muscle fibre revealed that fibres from cycloheximide-treated animals contained myofilaments, although there were some differences in myofilament structure between normal and treated animals. The formation of the muscle fibres in the absence of protein synthesis is discussed.     

Impact of Lauric Arginate Application Form on its Antimicrobial Activity in Meat Emulsions

Lauric arginate (LAE) is a food-grade cationic surfactant that is highly active against a wide range of food pathogens (Listeria monocytogenes, Salmonella, and Escherichia coli) and food spoilers (Lactobacilli, yeast, and molds). The antimicrobial efficacy of LAE in compositionally complex environments is likely to be negatively impacted by its interactions with food ingredients. Therefore, we investigated different application systems of LAE and their impact on its antimicrobial efficacy when added to “Lyoner style” sausages. LAE was applied as a powder, aqueous solution, in oil-in-water emulsions with different droplet sizes, and as solid lipid particles (SLP) with different droplet sizes. Structures of the systems were identified by optical microscopy, differential scanning calorimetry (DSC) and static light scattering. A recontamination on the surface of sliced sausages was simulated using Listeria innocua as the target organism (2 log colony forming units (CFU)/slice), and the antimicrobial impact of 1,000, 1,500 and 2,000 μg/g applied LAE in the sausage was examined by growth curves. A modeling of the CFU-time relationship was carried out to provide a better evaluation of the antimicrobial activity of LAE. Finally, we carried out an isothermal titration calorimetry (ITC) analysis to simulate the interactions between LAE and proteins in the sausage matrix. Results revealed that the application systems differed in their surface area and, therefore, showed different antimicrobial activities when incorporated into sausage. The study demonstrated that the SLP and emulsions as LAE application systems increased the antimicrobial activity against microbial growth on the surface of sliced “Lyoner style” sausages.