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Actinomyces viscosus cell-free synthesis of extracellular slime polysaccharide.

A cell-free extract of Actinomyces viscosus T14Av catalyzed the synthesis of extracellular N-acetylglucosamine-rich slime polysaccharide. The activity was localized in the cytoplasmic membrane fraction and required the presence of ADP-glucose and UDP-N-acetylglucosamine. Maximal activity was demonstrated at pH 7.5 and also required the presence of divalent cations such as Mg2+ or Mn2+. Extracellular slime appeared to serve as a primer for slime biosynthesis. The antibiotic tunicamycin acted as an inhibitor of slime formation. However, another glucosamine analogue, amphomycin, as well as the antibiotic bacitracin produced moderate stimulatory effects on slime biosynthesis.

Actinomyces↗

Susceptibility of Actinomyces israelii to antibiotics, sodium hypochlorite and calcium hydroxide.

Actinomyces israelii has been repeatedly implicated as a cause of failure of endodontic therapy. This study investigated the antimicrobial effect of antibiotics as well as intracanal medicaments, sodium hypochlorite solution and calcium hydroxide, on this important pathogen. Growth of A. israelii was inhibited by low concentrations of antibiotics, yet high concentrations were not bactericidal for A. israelii over 1 week. When A. israelii was exposed for 2-6 weeks at concentrations equivalent to clinical serum levels, the antibiotics were lethal. The results reveal a species-specific antibiotic tolerance for A. israelii. Both sodium hypochlorite solution and calcium hydroxide were found to be highly effective in killing A. israelii.

Actinomyces↗

Antimicrobial activity of varying concentrations of sodium hypochlorite on the endodontic microorganisms Actinomyces israelii, A. naeslundii, Candida albicans and Enterococcus faecalis.

AIM: To determine the resistance of microorganisms associated with refractory endodontic infections to sodium hypochlorite used as a root canal irrigant. METHODOLOGY: Two strains each of Actinomyces naeslundii, Candida albicans and Enterococcus faecalis were tested as late logarithmic phase inocula, against sodium hypochlorite adjusted to 0.5, 1.0, 2.5 and 5.25% w/v. Contact times used were 0, 10, 20, 30, 60 and 120 s. In the case of E. faecalis, additional experiments used contact times of 1.0, 2.0, 5.0, 10.0 and 30.0 min. Anti-microbial action was halted by sodium thiosulphate addition. Survivors were measured primarily using viable counts on drop plates. Additionally, pour plates were used to count low colony-forming units (cfu) and dilutions to 10(-6) were used to count high cfu. RESULTS: All concentrations of NaOCl lowered cfu below the limit of detection after 10 s in the case of A. naeslundii and C. albicans. However, E. faecalis proved to be more resistant to NaOCl. Using 0.5% NaOCl for 30 min reduced cfu to zero for both strains tested. This compares with 10 min for 1.0%, 5 min for 2.5% and 2 min for 5.25% (P < 0.001). Regression analysis for the dependent variable log(e)(count + 1) with log(e)(time + 1) and concentration as explanatory variables gave rise to a significant interaction between time and concentration (P < 0.001). CONCLUSION: The published association of E. faecalis with refractory endodontic infection may result, at least partially, from high resistance of this species to NaOCl. This does not appear to be the case with A. naeslundii or C. albicans.

Actinomyces↗

Rat memory T lymphocytes: in vitro proliferation induced by antigens of Actinomyces viscosus.

Peripheral T lymphocytes from RIC-Sprague-Dawley rats primed in vivo several months earlier with Actinomyces viscosus Ny 1 reacted with a strong proliferative response upon antigenic challenge in vitro. Two different antigen preparations from A. viscosus Ny 1, a broken cell supernatant (BCS) and an extracellular fraction (EXC) gave maximum responses as measured by the uptake of [3H]thymidine at a concentration of 10 microgram at day 4 of culture. Phytohaemagglutinin (PHA) used as a control provoked a maximum proliferation in the same order of magnitude. T lymphocytes from unprimed, germfree and conventional animals showed a similar stimulation to PHA, but only a marginal reaction to BCS, and were not at all activated by EXC. Evidence is thus presented for efficient removal of cells responding non-specifically to B-cell mitogens.

Actinomyces↗

Rat memory T lymphocytes. II. Differences in macrophage-dependent activation shown by Actinomyces viscosus antigens and by mitogens, using silica in vitro.

Protein-coated silica, a macrophage toxin, was used to assess the requirement for accessory cells in the induction of an in vitro proliferative response to (i) antigens from Actinomyces viscosus and (ii) the mitogens conconavalin A (Con A) and phytohaemagglutinin (PHA). T cells were obtained from RIC-Sprague-Dawley rats primed in vivo with A. viscosus Nyl by splenectomy and filtering the spleen cell suspensions through Degalan Ig-anti-IgG columns. In the presence of 100 microgram silica/ml during 4 days of culture, the proliferative response of T lymphocytes was not diminished. In contrast, when the T cells were precultured with silica for 24 h, washed, and subsequently cultured with the antigen fractions, antigen-induced proliferation was abolished. This procedure, however, had no influence on mitogen-induced proliferation was abolished. This procedure, however, had no influence on mitogen-induced T-cell activation. It is therefore concluded that the antigen-dependent anamnestic in vitro response (but not activation by mitogens) of rat T lymphocytes needs help from silica-sensitive macrophages.

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Inhibition of Actinomyces viscosus--Porphyromonas gingivalis coadhesion by trypsin and other proteins.

Protease activity is associated with the coadhesion of Actinomyces viscosus and Porphyromonas gingivalis. To try to distinguish whether the recognition/adhesion or degradative functions of proteases are more crucial for coadhesion, we determined the effect of trypsin and other purchased proteases and proteins on coadhesion when they were incorporated in the coadhesion assay buffer or when A. viscosus cells were pretreated with trypsin. Coadhesion was measured by the decrease in turbidity caused by the absorption of A. viscosus cells from aqueous suspension by P. gingivalis-coated hexadecane droplets. Pretreatment of A. viscosus with trypsin had no obvious effect on the kinetics of coadhesion. Likewise, trypsinization of A. viscosus failed to aid or enhance coaggregation by chemically induced, trypsin activity-deficient mutants of B. gingivalis. In contrast, incorporating trypsin in the buffer during the coadhesion assay yielded a concentration-dependent inhibition of coadhesion greater than the inhibition found with the same concentration of other proteases. Coadhesion was also impaired to a greater extent by similar wt/vol concentrations of nonproteolytic proteins (bovine serum albumin (BSA), defatted BSA, gelatin, and casein), by antisera against whole P. gingivalis cells and fimbriae, by preimmune serum, and by the amino acid arginine but not lysine. These findings suggest that the role of proteases in coadhesion is not solely to enzymatically "prime" A. viscosus for more avid coadhesion and that their role as potential protein or peptide seeking adhesins should be considered.

Actinomyces viscosus↗

Pathogenicity of Actinomyces israelii and Arachnia propionica: experimental infection in guinea pigs and phagocytosis and intracellular killing by human polymorphonuclear leukocytes in vitro.

Strains of Actinomyces israelii and Arachnia propionica, isolated from clinical cases of failed endodontic therapy, were examined for: (i) their ability to survive and establish themselves in the soft connective tissue that grew into subcutaneously implanted tissue cages in guinea pigs; (ii) cell-surface hydrophobicity; and (iii) phagocytosis and killing by human polymorphonuclear leukocytes (PMNs) under aerobic and anaerobic conditions. Bacteria were inoculated into the tissue cages in guinea pigs and the cage contents were retrieved after 1, 7, 14 and 21 d for culturing and light and electron microscopy. Both bacterial species showed substantial decline in the number of bacteria by day 7 after the inoculation. Thereafter, the A. israelii strain recovered and, by day 21, had started to increase in number. Light and electron microscopy revealed the formation of typical actinomycotic colonies. A. propionica, on the other hand, continued to decline in number during the entire period of experimental infection and did not form colonies. Both strains were hydrophobic, readily phagocytosed and were efficiently killed by human PMNs under aerobic and anaerobic conditions in vitro. These results suggest that the pathogenicity of A. israelii is due to its ability to establish characteristic cohesive colonies consisting of branching filamentous organisms that are enmeshed in an extracellular matrix. It seems that the organisms existing in such colonies can collectively evade destruction and elimination by host phagocytic cells, whereas in vitro suspensions of the bacteria are easily phagocytosed and efficiently killed by PMNs. With respect to A. propionica, further investigations are necessary to understand its pathogenicity.

Actinomyces↗

Stimulatory effect of bicarbonate on the glycolysis of Actinomyces viscosus and its biochemical mechanism.

The effects of bicarbonate on acid production by 4 human strains of Actinomyces viscosus were estimated under anaerobic conditions. The rate of acid production was accelerated by bicarbonate 3-4 times as much as that without bicarbonate. The analyses of intracellular glycolytic intermediates, NAD and NADH revealed a decrease in NADH:NAD ratio and an increase in the level of 3-phosphoglycerate in the cells when bicarbonate was present. Furthermore, when bicarbonate was available, malate dehydrogenase and fumarate reductase in the succinate pathway were expected to function as NADH-oxidizing enzymes in addition to lactate dehydrogenase. These observations indicate the efficient regeneration of NAD in the presence of bicarbonate. Thus, the stimulation of A. viscosus glycolysis by bicarbonate was thought to stem from the activation of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) by the decrease in the level of NADH, because NADH was a strong inhibitor of G3PDH in this microorganism.

Acetates↗

Reduction in adherence of Actinomyces viscosus after exposure to low-frequency acoustic energy.

The ability of low-frequency (200 Hz) acoustic energy to reduce the adherence of Actinomyces viscosus T14V to saliva-treated hydroxyapatite (SHA) disks was studied. An acoustic pressure range between 0 and 65 kPa and exposure durations between 0 and 8 min were used to study the levels necessary to significantly alter adherence. The effects of acoustic exposure on both bacteria in liquid and bacteria already adhering to SHA disks were studied. A modified enzyme-linked immunosorbent assay was used to assess bacterial adherence. For bacterial suspensions exposed prior to addition to SHA disks, it was found that reductions in adherence were greater for lower bacterial concentrations. Exposure of bacteria already adhering to SHA disks resulted in a decrease in adherence that was independent of the bacterial concentration and linearly related to the logarithm of the exposure duration. In addition to affecting adherence, acoustic energy also dispersed bacterial aggregates. Our results support the concept that low-frequency sonic energy applied orally may be of therapeutic value in reducing adherence and colonization of teeth by plaque bacteria.

Acoustic Stimulation↗

Fimbrial-mediated colonization of murine teeth by Actinomyces naeslundii.

Groups of mice fed diets high in sucrose or glucose were orally inoculated with 10(10), 10(9) or 10(8) colony-forming units of one of the following Actinomyces naeslundii strains possessing the type 1 (T1+) and/or the type 2 (T2+) fimbriae: T14VJ1 (T1+, T2+), 5519 (T1+), 5951 (T2+), and 147 (non-fimbriated). Ninety-six hours after inoculation their upper jaws were cultured to look at the implantation of each of these strains on the teeth. In mice fed a sucrose diet, regardless of the presence or absence of fimbriae, each bacterial strain colonized 100% of the mice at the highest inoculation doses of the infecting organism. But at a dose of 10(8), T14V-J1 was the only strain which colonized 100% (12/12) of the mice, 5519 colonized 10/11, 5951 colonized 9/11 and 147 colonized 7/11. These differences were not statistically significant. When mice were fed a high-glucose diet, 100% infection was achieved with strains T14V-J1, 5519 and 5951 only at the highest dose of 10(10) colony-forming units. Strain 147 colonized in 8/9 of the mice at that dosage. At lower dosages, no bacterial strain implanted in 100% of the mice. In the glucose experiment at a dose of 10(8), strains expressing the T1 fimbriae implanted significantly better than strains without the T1 fimbriae. At a dose of 10(9) colony-forming units, the parent strain T14V-J1 implanted significantly better than strains without the T1 fimbriae. Similarly, strain 5519 (T1+) implanted significantly better than 5951 and implanted better than 147, although the difference was not significant. These results suggest that while the presence of the T1 and T2 fimbriae may confer some advantage in the establishment of these organisms in vivo, even the strains without fimbriae were able to colonize. Strains T14VJ1 and 5519 were found to bind well to hydroxyapatite treated with mouse saliva, while strains 5951 and 147 did not. Only T2 fimbriated strains T14V-J1 and 5951 exhibited a lactose-reversible coaggreation with indigenous strains of enterococci that may contribute to the elevated levels of colonization of strain 5951 in vivo.

Actinomyces↗

Mechanisms of inhibition by fluoride of urease activities of cell suspensions and biofilms of Staphylococcus epidermidis, Streptococcus salivarius, Actinomyces naeslundii and of dental plaque.

BACKGROUND/AIMS: Fluoride is known to be a potent inhibitor of bacterial ureases and can also act in the form of hydrofluoric acid as a transmembrane proton conductor to acidify the cytoplasm of intact cells with possible indirect, acid inhibition of urease. Our research objectives were to assess the inhibitory potencies of fluoride for three urease-positive bacteria commonly found in the mouth and to determine the relative importance of direct and indirect inhibition of ureases for overall inhibition of intact cells or biofilms. METHODS: The experimental design involved intact bacteria in suspensions, mono-organism biofilms, cell extracts, and dental plaque. Standard enzymatic assays for ammonia production from urea were used. RESULTS: We found that ureolysis by cells in suspensions or mono-organism biofilms of Staphylococcus epidermidis, Streptococcus salivarius or Actinomyces naeslundii was inhibited by fluoride at plaque levels of 0.1-0.5 mm in a pH-dependent manner. The results of experiments with the organic weak acids indomethacin and capric acid, which do not directly inhibit urease enzyme, indicated that weak-acid effects leading to cytoplasmic acidification are also involved in fluoride inhibition. However, direct fluoride inhibition of urease appeared to be the major mechanism for reduction in ureolytic activity in acid environments. Results of experiments with freshly harvested supragingival dental plaque indicated responses to fluoride similar to those of S. salivarius with pH-dependent fluoride inhibition and both direct and indirect inhibition of urease. CONCLUSION: Fluoride can act to diminish alkali production from urea by oral bacteria through direct and indirect mechanisms.

Actinomyces↗

Role of CD4 and CD8 T-cells in the induction of oral tolerance to Actinomyces viscosus in mice.

Mucosal presentation of Actinomyces viscosus results in the induction of antigen specific systemic suppressor cells in mice. The aim of the present study was to determine the phenotype of the suppressor cells responsible for the induction of oral tolerance to low doses of A. viscosus. When CD8 cell-depleted DBA/2 mice were intragastrically immunized and systemically immunized with A. viscosus, the delayed type hypersensitivity response was suppressed but not the levels of antigen specific serum antibodies. Adoptive transfer of orally tolerized CD4(+) cells to CD4(+)-depleted mice resulted in suppression of delayed type hypersensitivity response but not of the levels of antigen specific serum antibodies. In contrast, adoptive transfer of orally immunized CD8(+) cells to CD8(+)-depleted mice resulted in partially suppressed delayed type hypersensitivity response but significantly inhibited the levels of antigen specific serum antibodies. When orally tolerized CD8(+) cells were cocultured with systemically immunized CD8(+) cell-depleted spleen cells, splenic specific antibodies were inhibited. However, no suppression of splenic specific antibodies could be observed in the cultures containing orally tolerized CD4(+) cells and systemically immunized CD4(+) cell-depleted spleen cells. The results of the present study suggest that oral tolerance of humoral and cellular immunity induced by low doses of A. viscosus may be mediated by CD8(+) and CD4(+) cells, respectively.

Actinomyces viscosus↗

The role of CD4+ and CD8+ T cells on antibody production by murine Peyer's patch cells following mucosal presentation of Actinomyces viscosus.

The aim of this study was to determine the role of CD4 and CD8 cells on specific antibody production by murine Peyer's patch (PP) cells after oral immunization with Actinomyces viscosus in mice. Female DBA/2 mice were orally immunized with three low doses of heat-killed A. viscosus. Sham-immunized mice served as a control group. Mice were depleted of CD4 or CD8 cells by intraperitoneal injection of anti-CD4 or anti-CD8 antibodies daily for 3 days before oral immunization. One week after the last oral immunization, PPs were removed and cell suspensions were cultured with A. viscosus. Specific antibody production in the culture supernatants was assessed by enzyme-linked immunosorbent assay. The results showed that oral immunization with A. viscosus induced a predominant specific immunoglobulin A (IgA) response by PP cells and, to a lesser extent, IgM antibodies. Depletion of CD4 but not CD8 cells suppressed the production of specific antibodies. These results suggest that oral immunization with low doses of A. viscosus may induce the production of specific antibodies by murine PP cells in a CD4-cell-dependent fashion.

Actinomyces viscosus↗

Cell surface hydrophobicity of Actinomyces pyogenes determined by hexadecane adherence- and salt aggregation studies.

Cell surface hydrophobicities of Actinomyces pyogenes were determined by measuring the adherence of the bacteria to hexadecane droplets and by salt aggregation tests. Among 42 A. pyogenes cultures tested 25 (60%) adhered strongly (adherence greater than or equal to 75%) and 17 (40%) less pronounced (adherence between 25-75%) to the hexadecane droplets. Pre-treatment of the bacteria with proteolytic enzymes completely eliminated the adherence properties whereas heat treatment had no effect. The salt aggregation studies revealed that 4 (10%) cultures aggregated in ammonium sulfate solutions of a molarity of 0.05 mol/l, 5 (12%), 14 (33%) and 3 (7%) cultures in ammonium sulfate solutions with molarities of greater than or equal to 1.5 mol/l, greater than or equal to 3 mol/l and greater than or equal to 4.5 mol/l, respectively. No aggregation at all could be observed with 16 (38%) of the cultures. Pronase treatment completely eliminated the salt aggregation reactions, trypsin- and heat treatment had no effect. The results from hexadecane adherence and salt aggregation did not correspond. The differences in surface hydrophobicities, possibly related to adherence properties of A. pyogenes, could be used for epidemiological typing of individual cultures of this bacterial species.

Actinomyces↗

Evaluation of the API Coryne test system for identification of Actinomyces pyogenes.

The present study was designed to evaluate the accuracy of the API Coryne test system for identification of Actinomyces pyogenes. The test system correctly identified 36 of 42 A. pyogenes and 4 of 5 comparatively studied Arcanobacterium haemolyticum-cultures. The biochemical profiles of the remaining 6 A. pyogenes- and 1 A. haemolyticum-cultures were not included in the analytical profile index. None of the cultures were misidentified. According to the API database (ATB Plus V 1.5.4.) the unidentified cultures could be correctly identified as A. pyogenes and A. haemolyticum respectively. A greater repertoire of A. pyogenes specific biochemical profiles incorporated into the analytical profile index would improve the applicability of this test system for veterinary diagnostics.

Actinomyces↗

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.

Actinomyces↗

Purification and further characterization of a haemolysin of Actinomyces pyogenes.

A haemolysin produced by Actinomyces pyogenes ATCC 8164 was purified from culture supernatant by ammonium sulphate and polyethylene glycol precipitation, ion-exchange chromatography on DEAE-Sephacel, and fast-protein-liquid-chromatography on Superose 12 prep grade. The purified haemolysin, designated as pyolysin, displayed a single band on poly-acrylamide gel electrophoresis, indicating a molecular weight of 55000. Additionally, using gel filtration, the same molecular weight was estimated. Further studies of the eluate of ion-exchange chromatography using isoelectric focusing also revealed a single protein band at pH 9.38 with haemolytic activity. A specific antiserum produced against pyolysin inhibited the haemolytic activity. The purity of the isolated protein was also determined by Western Blot analysis with antiserum obtained from a cow inoculated with culture supernatant from A. pyogenes and Peptococcus indolicus. The isolated pyolysin appeared to be heat-labile and displayed cytotoxic effects on poly-morphonuclear leucocytes and on pTK2 kidney cells.

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Further characterization of alpha 2-macroglobulin binding properties of Actinomyces pyogenes.

Binding of 125I-labelled alpha 2-macroglobulin (alpha 2M) to Actinomyces pyogenes was investigated. The binding of alpha 2M proved to be time dependent, saturable, and could be inhibited by unlabelled alpha 2M, but not by fibrinogen, haptoglobin, fibronectin, or albumin. The alpha 2M binding site was sensitive to treatment with proteolytic enzymes and heat, indicating its protein nature. The dissociation constant (Kd) was 4.447 x 10(-9) M, and the number of binding sites per bacterial cell was calculated to be 5200. The kinetic analysis indicated an homogeneous population of binding sites. Binding of alpha 2M to the surface of A. pyogenes had no significant influence on the phagocytosis of the bacteria by polymorphonuclear leucocytes.

Actinomyces↗