Antibiofilm efficacy of photosensitizer-functionalized bioactive nanoparticles on multispecies biofilm.

INTRODUCTION: Newer disinfection strategies based on antibacterial nanoparticles and photodynamic therapy (PDT) aim to eliminate residual biofilm bacteria during root canal treatment. The aim of the current study was to test the newly developed rose bengal-functionalized chitosan nanoparticles (CSRBnps) for their interaction/uptake with monospecies bacteria/biofilm and assess their antibiofilm efficacy on a multispecies biofilm model in vitro. METHODS: The interaction of CSRBnps with bacterial cells was conducted using atomic force microscopy. Their membrane-damaging effect was determined by measuring the absorbance at 260 nm (OD260nm) using Enterococcus faecalis. The penetration of CSRBnps into E. faecalis biofilms was evaluated using confocal laser scanning microscopy (CLSM). Multispecies biofilms of Streptococcus oralis, Prevotella intermedia, and Actinomyces naeslundii were grown on dentin sections for 21 days to assess the antibiofilm efficacy. The biofilms were subjected to PDT (60 J/cm(2)) using CSRBnps and rose bengal. The treated/untreated biofilms were examined under scanning electron microscopy and CLSM. RESULTS: The CSRBnps synthesized were 60 ± 20 nm and showed absorption spectra similar to rose bengal. Atomic force microscopy showed adherence of CSRBnps to bacteria, roughening of cell surface, and cell disruption after PDT. CSRBnp treatment resulted in significantly increased bacterial membrane damage (P < .05). CSRBnps exhibited deeper penetration into the biofilm structure. Scanning electron microscopy and CLSM confirmed the complete disruption of multispecies biofilm with a reduction in viable bacteria and biofilm thickness (P < .05). CONCLUSIONS: These novel photosensitizer functionalized bioactive nanoparticles with increased affinity to bacterial cell membrane, higher penetration into biofilm structure, and enhanced ability to eliminate clinically relevant multispecies bacterial biofilm present a potential antibiofilm agent for root canal disinfection.

Effects of short-chain fatty acids on Actinomyces naeslundii biofilm formation.

Actinomyces naeslundii is an early colonizer and has important roles in the development of the oral biofilm. Short-chain fatty acids (SCFA) are secreted extracellularly as a product of metabolism by gram-negative anaerobes, e.g. Porphyromonas gingivalis and Fusobacterium nucleatum; and the SCFA may affect biofilm development with interaction between A. naeslundii and gram-negative bacteria. Our aim was to investigate the effects of SCFA on biofilm formation by A. naeslundii and to determine the mechanism. We used the biofilm formation assay in 96-well microtiter plates in tryptic soy broth without dextrose and with 0.25% sucrose using safranin stain of the biofilm monitoring 492 nm absorbance. To determine the mechanism by SCFA, the production of chaperones and stress-response proteins (GrpE and GroEL) in biofilm formation was examined using Western blot fluorescence activity with GrpE and GroEL antibodies. Adding butyric acid (6.25 mm) 0, 6 and 10 h after beginning culture significantly increased biofilm formation by A. naeslundii, and upregulation was observed at 16 h. Upregulation was also observed using appropriate concentrations of other SCFA. In the upregulated biofilm, production of GrpE and GroEL was higher where membrane-damaged or dead cells were also observed. The upregulated biofilm was significantly reduced by addition of anti-GroEL antibody. The data suggest biofilm formation by A. naeslundii was upregulated dependent on the production of stress proteins, and addition of SCFA increased membrane-damaged or dead cells. Production of GroEL may physically play an important role in biofilm development.

Antimicrobial activity of ethanol extracts of Laminaria japonica against oral microorganisms.

Laminaria japonica is a brown alga, which is consumed widely in Korea, Japan, and China. This study investigated the antimicrobial activity of ethanol extracts of L. japonica against oral microbial species to assess the possible application of L. japonica extracts in dental care products. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined in culture medium using a microdilution method. The MICs of ethanol extracts of L. japonica with oral streptococci were 62.5-500 µg/ml and the MBCs were 125-1000 µg/ml. The MICs of Actinomyces naeslundii and Actinomyces odontolyticus were 250 and 62.5 µg/ml, respectively. The MBCs of A. naeslundii and A. odontolyticus were 500 and 250 µg/ml, respectively. The MICs were 250 and 62.5 µg/ml for Fusobacterium nucleatum and Porphyromonas gingivalis, respectively. The killing of Streptococcus mutans and P. gingivalis was dependent on the incubation time. The killing of S. mutans, A. odontolyticus, and P. gingivalis was significantly dependent on the extract concentration. Bacterial treatment with L. japonica extracts changed the cell surface texture of S. mutans, A. odontolyticus, and P. gingivalis. The results of this study suggest that L. japonica extracts may be useful for the development of antimicrobial agents to combat oral pathogens.

Effect of a novel antimicrobial peptide chrysophsin-1 on oral pathogens and Streptococcus mutans biofilms.

Dental caries and pulpal diseases are common oral bacterial infectious diseases. Controlling and reducing the causative pathogens, such as Streptococcus mutans and Enterococcus faecalis, is a key step toward prevention and treatment of the two diseases. Chrysophsin-1 is a cationic antimicrobial peptide having broad-spectrum bactericidal activity against both Gram-positive and Gram-negative bacteria. In this study, we investigated the antibacterial activity of chrysophsin-1 against several oral pathogens and S. mutans biofilms and performed a preliminary study of the antimicrobial mechanism. Cytotoxic activity of chrysophsin-1 against human gingival fibroblasts (HGFs) was investigated. Minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and time-kill assay were used to evaluate the killing effect of chrysophsin-1. Scanning electron microscopy (SEM) was used to analyze morphological and membrane change in oral pathogens. Live/Dead staining, in conjunction with confocal scanning laser microscopy (CSLM), was used to observe and analyze S. mutans biofilms. MIC and MBC results demonstrated that chrysophsin-1 had different antimicrobial activities against the tested oral microbes. Lysis and pore formation of the cytomembrane were observed following treatment of the bacteria with chrysophsin-1 for 4h or 24h by SEM. Furthermore, CLSM images showed that chrysophsin-1 remarkably reduced the viability of cells within biofilms and had a significantly lethal effect against S. mutans biofilms. Toxicity studies showed that chrysophsin-1 at concentration between 8 µg/ml and 32 µg/ml had little effect on viability of HGFs in 5 min. Our findings suggest that chrysophsin-1 may have potential clinical applications in the prevention and treatment of dental caries and pulpal diseases.

Actinomycosis of the jaws--histopathological study of 45 patients shows significant involvement in bisphosphonate-associated osteonecrosis and infected osteoradionecrosis.

Actinomycosis of the jaws is a rare disease, which has been recently described in patients with infected osteoradionecrosis (IORN) and bisphosphonate-associated osteonecrosis (BON). We investigated our archive material for Actinomycosis of the jaws with special regard to underlying disease. Out of a total number of 45 patients with Actinomycosis, 43 (93.5%) suffered from BON (58.7%) or IORN (35.6%), while there were only 3 patients (6.7%) without anti-tumor treatment. In all cases, we found direct association of Actinomyces colonies with bone; in the surrounding medullary space, mixed inflammatory infiltrates with variable amounts of osteoclasts were a typical finding. Pseudoepitheliomatous hyperplasia occurred in 60.9% of patients. Cell-rich vessel obliteration was seen in less than 25.9% of BON patients, while hyalinized vessel obliteration was obtained in 37.5% of IORN patients. Additionally performed polymerase chain reaction (PCR) on paraffin-embedded and ethylene diamine tetracetic acid (EDTA)-decalcified tissue specimens confirmed the presence of Actinomyces israelii in seven of seven cases analyzed. We conclude that Actinomycosis of the jaws is a particular complication in patients with BON and/or IORN. Patients with Actinomycosis of the jaws during or after these forms of anti-cancer therapy are suggested to represent a distinct patient cohort with a relevant impairment of their general condition.

Direct detection of cell surface interactive forces of sessile, fimbriated and non-fimbriated Actinomyces spp. using atomic force microscopy.

Actinomyces species are predominant early colonizers of the oral cavity and prime mediators of inter-bacterial adhesion and coaggregation. Previous workers have evaluated the adhesion of Actinomyces spp. by quantitative assessment of sessile, as opposed to planktonic cells attached to substrates, but did not quantify the cell surface interactive forces. Therefore we used atomic force microscopy to directly detect the interactive force between an approaching silicon tip and sessile Actinomyces spp. adhering to a substrate, at nanonewton (nN) range force levels. A total of eight strains each belonging to fimbriated and non-fimbriated Actinomyces species were employed, namely A. bovis, A. gerencseriae, A. israelii, A. meyeri, A. naeslundii genospecies 1 and 2, A. odontolyticus and A. viscosus. The sterile mica discs, used as the adhesion substrate, were immersed in mono-species bacterial suspensions for five days to obtain a thin bacterial biofilm. Interactive forces were measured using a silicon nitride cantilever attached to a Nanoscope IIIA atomic force microscope. The interactive forces between the approaching silicon nitride tip and bacterial biofilm surfaces were randomly quantified at three different locations on each cell; namely, the cell surface proper, the periphery of the cell and the substrate and, the interface between two cells. When the interactive forces at these locations of the same species were compared, significantly higher force levels at the cell-cell interface than the other two locations were noted with A. gerencseriae (P < 0.001), A. viscosus (P < 0.01) and A. israelii (P < 0.05). When the interactive forces of different Actinomyces spp. at an identical location were compared, fimbriated A. naeslundii genospecies 2 showed the greatest interactive force at the cell surface proper (-32.6 +/- 8.7 nN, P < 0.01). A. naeslundii genospecies 1, 2 and A. viscosus demonstrated greater interactive force at the cell-mica periphery than the other five species (P < 0.05); A. viscosus (-34.6 +/- 10.5 nN) displayed greater interactive force at the cell-cell interface than the others (P < 0.01), except for A. gerencseriae (P > 0.05). These data indicate that fimbriated Actinomyces spp., including A. naeslundii genospecies 1, 2 and A. viscosus exert higher cell surface interactive forces than those devoid of fimbriae and, such variable force levels may modulate their adhesion and coaggregation during biofilm formation.

Copper accumulation in actinomyces druses during endometritis after long-term use of an intrauterine contraceptive device.

A 32-year-old woman carried a copper intrauterine contraceptive device (IUCD) or intrauterine pessar (IAP) for more than 5 years. She had acyclic menstrual bleedings and underwent a corpus abrasio after explantation of IUCD. The histological study of paraffin sections showed an actinomycotic endometritis with brown to black deposits in or around typical actinomyces druses, but there was no carcinoma. The electron microscopic study of these accumulations by electron energy loss spectroscopy (EELS) in TEM demonstrated copper deposits in the shell and matrix of these druses as well as inside the bacteria. With scanning electron microscopy (SEM) and Energy Dispersive X-Ray Analysis (EDX), the electron-dense accumulations revealed high signals for copper and sulfur, but also of phosphorus and oxygen in a lower extent. This copper accumulation is discussed as an active uptake and concentration by these actinomyces bacteria.

Oral cytology in cannabis smokers.

The effects of cannabis/methaqualone/tobacco smoking on the epithelial cells of the tongue, buccal mucosa and floor of the mouth were examined. Oral mucosal smears for detection of cellular changes were taken from 4 sites in 16 patients. The tongue blade scraping technique was used. The sites sampled included the buccal mucosa (left and right sides), the posterior dorsum of the tongue and the anterior floor of the mouth. Tobacco smoking and non-smoking controls were also examined. The only significant difference between cannabis users and controls was the greater prevalence of bacterial cells in the smears taken from cannabis users. However, there were also greater numbers of degenerate and atypical squamous cells in cannabis smokers than in cigarette-smoking and non-smoking controls. Epithelial cells in smears taken from cannabis users and tobacco-smoking controls also showed koilocytic changes, which were not seen in smears taken from non-smoking controls. Koilocytosis is indicative of human papilloma virus infection, although no apparent lesions were seen in the patients from whom smears had been taken. It would appear that there is a greater tendency towards damaged and immature surface epithelial cells in cannabis smokers.

Molecular strategies for fimbrial expression and assembly.

Fimbriae or pili are long, filamentous, multimeric macromolecules found on the bacterial cell surface. Bacteria express a diverse array of fimbriae or pili that are involved in bacterial adherence and invasion. Fimbriae can be categorized based on their modes of expression and assembly. Type I fimbriae and P pili are distributed peritrichously and translocated to the cell surface by a chaperone/usher pathway. Type 4 pili are located at the pole of the cell and assembled via the type II secretion system. Curli fimbriae are coiled surface structures assembled by an extracellular nucleation/precipitation pathway. Fimbriae of oral gram-negative and gram-positive bacteria have not been well-studied as compared with the fimbriae of enteric pathogens. Oral pathogens, such as Eikenella corrodens, Actinobacillus actinomycetemcomitans, and Porphyromonas gingivalis, possess fimbriae that have been implicated in bacterial adhesion and invasion. These fimbriae are potential virulence factors in oral infectious processes. A. actinomycetemcomitans and E. corrodens have Type 4-like fimbriae, whereas P. gingivalis displays a unique type of fimbriae. To date, fimbriae of the oral primary colonizers, Actinomyces naeslundii and Streptococcus parasanguis, represent the only fimbriae characterized for any gram-positive bacteria. The putative major fimbrial subunits, FimA and FimP of A. naeslundii and Fap1 of S. parasanguis, contain a signal sequence and cell-wall-sorting signal. The presence of extensive dipeptide repeats in Fap1 makes it unique among fimbrial molecules. Based on experimental data, a nucleation/precipitation pathway is proposed for fimbrial biogenesis of both S. parasanguis and A. naeslundii, although we cannot rule out an alternative covalent linkage model. The model systems described in this review served as a framework for hypotheses for how the known molecular factors of fimbriae on oral bacteria may be expressed and assembled.

Actinomyces infection in porous polyethylene orbital implant.

BACKGROUND: A 60-year-old patient developed actinomycotic inflammation within a porous polyethylene orbital implant which she received following enucleation. METHODS: She had repeated conjunctival exposures with inflammation; the primary implant was removed and replaced with another one. RESULTS: The anterior two-thirds of the porous implant was infiltrated with numerous actinomycotic granules surrounded by polymorphonuclear cells and necrotic debris. The organisms were demonstrated with Gram stains on the histopathologic preparations and with scanning electron microscopy. Within the zones of inflammation, the polyethylene skeleton of the implant was extensively damaged. CONCLUSION: Actinomycetes have been described as causative organisms in conjunctivitis, blepharitis, canaliculitis, dacryocystitis and keratitis, but to the best of our knowledge actinomycotic involvement has never been reported in an infected porous orbital implant.

Cell surface structures of Actinomyces israelii.

Actinomyces israelii is the most common cause of human actinomycosis, a chronic granulomatous infection. Periapical actinomycosis involving A. israelii has been identified as an important cause of failure of conventional endodontic treatment. Structures on the bacterial cell surface have been implicated in the pathogenicity of Actinomyces. In this study the ultrastructure of A. israelii was investigated by electron microscopy. Negatively stained preparations revealed the presence of hairlike fimbriae protruding through a thick surface coat on some species, whilst thin sectioning disclosed a Gram-positive cell wall surrounded by a fuzzy outer coat. These structures may be important for the pathogenicity of A. israelii.

A new genus of the order Actinomycetales, Spirilliplanes gen. nov., with description of Spirilliplanes yamanashiensis sp. nov.

Actinomycete strain YU127-1T (T = type strain), which produces zoospores, was isolated from a soil sample. The aerial mycelium of this organism at maturity forms short chains of spores. The hyphae form coils, and sporangia are not observed. Strain YU127-1T contains glutamic acid, glucosamine, glycine, alanine, and meso-diaminopimelic acid in its cell wall (wall chemotype II), 3-O-methylmannose, mannose, xylose, and glucose as whole-cell sugars, meanaquinone 10(H4), and glycolyl cell wall polysaccharides and has a guanine-plus-cytosine content of 69.0 mol%. Mycolic acids are absent. Phosphatidylinositol and phosphatidylethanolamine are diagnostic phospholipids. The chemotaxonomic data, except for the lack of arabinose in the whole-cell sugars, indicate that this strain belongs to the family Micromonosporaceae. The morphological and physiological characteristics and chemotaxonomic and phylogenetic data for this strain differ from those of the previously described actinomycetes. We therefore propose a new genus, spirilliplanes, for this organism; the type species of the genus is Spirilliplanes yamanashiensis sp. nov., and the type strain of S. yamanashiensis is strain YU127-1 (= IFO 15828).

Characterization of fibrinogen-binding properties of Actinomyces pyogenes.

This study was designed to further characterize the fibrinogen-binding properties of Actinomyces pyogenes. The fibrinogen-binding capacities of a selected A. pyogenes culture could be significantly enhanced by heat pretreatment (60 degrees C, 1 h) of the bacteria. The fibrinogen-binding site seemed to be a protein which was specific for fibrinogen. In phagocytosis studies, binding of fibrinogen to A. pyogenes significantly increased the phagocytic capacity of bovine polymorphonuclear leucocytes.

[Pseudo-sulfur granules (pseudo-actinomycotic granules) in intrauterine device patients]. / Pseudosulfurgranula (Pseudoaktinomyzesdrusen) bei Intrauterinpessar-Trägerinnen.

In 14 tissues from uterine curettages from women with intrauterine contraceptive devices (IUD) we found structures were discovered, which could easily be mistaken for actinomycotic sulphur granules. These were characterised by radial filamentous eosinophilic structures with peripheral club-like swellings. The dissimilarities to a true sulphur granule are evident with Gram staining which shows single filaments substantially wider than those of the true actinomycotic granules. Until now the nature of the described structures is not clear. They are possibly deposits on the copper thread of the IUD.

An in vitro model for adhesion of bacteria to human tooth root surfaces.

A model mimicking bacterial colonization of dentine has been developed. It employs uniform particles of pulverized human tooth root tissue incubated with radioactively labelled bacteria. After incubation, the number of attaching bacteria is quantified. Attachment of Streptococcus mutans UA140, Actinomyces viscosus T14, and Lactobacillus casei 101 was found to be time dependent and complete within 1-3 h. Dissociation constants (Kd) of the interactions equalled 2.5 x 10(8) and 1.6 x 10(8) cells/ml, for Strep. mutants and A. viscosus, respectively. The Kd for L. casei could not be determined as attachment was not saturable. The putative tissue components involved in adherence were studied by determining the attachment of bacteria in the presence of competing strains. The results suggest that Strep. mutans and A. viscosus recognized and competed for the same ligand (probably collagen) in the dentine. L. casei attachment did not complete with the attachment of Strep. mutans and A. viscosus. Attachment of all strains was modified by preincubation with saliva and varied with bacterial strain and saliva donor.

Actinomycosis of the neck: diagnosis by fine-needle aspiration biopsy.

A patient with actinomyces infection of the posterior neck was diagnosed by fine-needle aspiration biopsy. The lesion presented as a recurrent, firm, and indurated mass that was clinically diagnosed as adenitis and cellulitis and was thought to be a lymphoma 6 months after the onset of his illness. Smears and cell block sections of the aspirate showed characteristic colonies ("sulfur granules") of actinomyces. Subsequent regional lymph node biopsy revealed reactive lymphoid hyperplasia.

Fibronectin staining detects micro-organisms in aged and Alzheimer's disease brain.

Filamentous, fibronectin-immunopositive structures, previously described in Alzheimer's disease and control brains were negative for neuronal, glial, and macrophage markers. The present study sought to determine the nature of these entities and to further characterize their morphology, immunoreactivity and distribution between neuropathologies. Ultrastructural analysis shows these formations to be filamentous micro-organisms, which may belong to the actinomycetes. Immunohistochemistry for the cell-stress protein ubiquitin is consistently positive in these organisms. They are also present in Down's syndrome, dementia pugilistica, amyotrophic lateral sclerosis with dementia, and Parkinson's disease. The pattern of tissue distribution implies a pre-mortem invasion of the brain, and, as the micro-organism is present at a four to five-fold higher frequency in Alzheimer's disease, it may act pathogenically in this dementing illness.