Actinomyces georgiae sp. nov., Actinomyces gerencseriae sp. nov., designation of two genospecies of Actinomyces naeslundii, and inclusion of A. naeslundii serotypes II and III and Actinomyces viscosus serotype II in A. naeslundii genospecies 2

DNAs of type strains and representative members of Actinomyces groups from the human periodontal flora and from other habitats were compared by using the S1 nuclease procedure to determine their genetic relatedness. One rather common group from the human periodontal flora, previously called "Actinomyces D08," is phenotypically distinct from, and genetically unrelated to, previously described species. We propose the name of Actinomyces georgiae for this organism; the type strain is strain ATCC 49285. Another common group from the human periodontal flora is Actinomyces israelii serotype II, which was found genetically distinct from the type strain of A. israelii (serotype I) and from other previously described species of Actinomyces. We propose the name Actinomyces gerencseriae for this organism; the type strain is strain ATCC 23860. A. naeslundii serotype I strains were distinct from the other strains studied. A separate genospecies which included strains of A. naeslundii serotypes II and III and A. viscosus serotype II was delineated. Strains of Actinomyces serotype WVA 963 constitute an additional distinct genospecies. Because there are no reliable phenotypic tests, other than serological analyses, to differentiate Actinomyces serotype WVA 963 and the two genospecies of A. naeslundii, no taxonomic changes are proposed for these three genospecies.     

Transformation of Actinomyces spp. by a gram-negative broad-host-range plasmid.

The gram-negative broad-host-range vector pJRD215 was transferred by electroporation into strains of Actinomyces viscosus or Actinomyces naeslundii at efficiencies which ranged from 10(2) to 10(7) transformants per microgram of plasmid DNA. The Actinomyces transformants expressed pJRD215-encoded resistance to kanamycin and streptomycin. Moreover, the transforming plasmid DNA had not undergone any deletions or rearrangements, nor had it integrated into the genomes of these strains.     

Aggregation of Actinomyces strains by extracellular vesicles produced by Bacteroides gingivalis

The aggregation of Actinomyces viscosus and Actinomyces naeslundii with extracellular vesicles of Bacteroides gingivalis was studied. Factors influencing the aggregation phenomenon were examined. L-Arginine was found to effectively inhibit aggregation as was an antibody preparation directed against a B. gingivalis surface hemagglutinin. Aggregation occurred over a wide pH range and did not seem to be affected by high salt concentrations or the presence of carbohydrates. Treatment of the vesicle preparation with proteases, sodium dodecyl sulphate, and high temperatures diminished or eliminated aggregation, while similar treatment of the Actinomyces had no effect on aggregation.     

Isolation,partial purification and preliminary characterization of a bacteriocin from Streptococcus mutans Rm-10

An extracellular bactericidal substance was isolated from the supernatant of Streptococcus mutans Rm-10 culture fluid and partially purified with 60% ammonium sulfate precipitation, differential centrifugation, and gel filtration on Sephadex G-200. There was a good correlation of the sensitivity profiles of indicator strains whether assayed on solid medium or with purified material from cell-free culture fluid, indicating that the same inhibitory substance is produced on solid medium and in broth. Vapor from organic solvents such as chloroform, acetone, ethanol, and ether as well as heat treatment at 100 degrees C for 30 min had little effect on the bactericidal factor. It was sensitive to trypsin and pronase and resistant to deoxyribonuclease, ribonuclease, lysozyme, and phospholipase C. The inhibitor was not infective, and electron microscopic studies failed to reveal phage or phage-like particles in concentrated solutions of the bactericidal material. The results indicate that the extracellular bactericidal substance is indeed a bacteriocin. Activity in broth cultures reached a maximum only after exponential growth had ceased. It was active against other streptococcal strains as well as strains of Actinomyces naeslundii, A. viscosus, Bacillus subtilis and Staphylococcus aureus, but not against strains of Fusobacterium nucleatum and Escherichia coli.     

Coaggregation between Prevotella nigrescens and Prevotella intermedia with Actinomyces naeslundii strains

Abstract Using a visual coaggregation assay, 43% (6 of 14) of Prevotella nigrescens and 50% (4 of 8) of Prevotella intermedia strains coaggregated with Actinomyces naeslundii strains which represented the six Actinomyces coaggregation groups (A to F). For both species, coaggregation occurred most frequently with A. naeslundii strains from coaggregation groups C, D and E. No coaggregation was observed with Actinomyces israelii , Actinomyces odontolyticus or six oral Streptococcus species. Coaggregation was not inhibited by lactose, saliva or serum. Pretreatment of Prevotella strains with heat, SDS and proteinase K abolished coaggregation when the treated cells were added to untreated Actinomyces strains. The same pretreatment of the Actinomyces strains had no effect on their ability to coaggregate with untreated Prevotella strains. Pretreatment of all coaggregating P. nigrescens strains with trypsin abolished coaggregation, whereas the coaggregation ability of the P. intermedia and Actinomyces strains was resistant to trypsin pretreatment. Pretreatment of the strains of both Prevotella species and the Actinomyces with periodate abolished coaggregation in all cases. These results suggest that the Prevotella strains each possess a protein coaggregation adhesin, which for the P. intermedia strains is resistant to trypsin, that interacts with a non-protein receptor on the A. naeslundii strains.     

Detection and localization of a lectin on Actinomyces viscosus T14V by monoclonal antibodies

A cell-associated lectin activity that mediates lactose-inhibitable adherence of Actinomyces viscosus T14V has been localized to a specific population of fimbriae by the use of monoclonal antibodies. Nine monoclonal antibodies were produced that reacted with only 1 of 2 immunoelectrophoretically distinct fimbrial components on T14V. The fibrillar morphology of this component was revealed by the immunoelectronmicroscopic examination of bacteria incubated with the monoclonal antibodies. The lectin activity associated with these structures was detected when isolated fimbriae were cross-linked with monoclonal antibodies to form immune complexes with agglutination activity for neuraminidase-treated human erythrocytes, a reaction that was inhibited by lactose. Although the 9 monoclonal antibodies differed in their fine specificities, they reacted only with strains of A. viscosus and A. naeslundii that exhibited lactose-inhibitable adherence. These findings indicate that the lectin activity common to these bacteria resides on fimbriae that are antigenically related to those of T14V.     

Effect of <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis> Vesicles on Coaggregation of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> to Oral Microorganisms

Vesicles from the outer membrane of Porphyromonas gingivalis have the ability to aggregate a wide range of Streptococcus spp., Fusobacterium nucleatum, Actinomyces naeslundii, and Actinomyces viscosus. We found that in the presence of P. gingivalis vesicles, Staphylococcus aureus coaggregated with Streptococcus spp., and the mycelium-type Candida albicans, but not the yeast type. Autoaggregation of S. aureus in the presence of P. gingivalis vesicles is inhibited by L-arginine, L-lysine, and L-cysteine. Both the methicillin-sensitive (MSSA) and -resistant (MRSA) strains of S. aureus were able to coaggregate with Streptococcus spp., A. naeslundii, and A. viscosus when they were treated with P. gingivalis vesicles. P. gingivalis vesicle-treated mycelium-type C. albicans coaggregated with S. aureus, but the yeast-type did not. These results indicate that strains of S. aureus, including MRSA, could adhere to oral biofilms in dental plaque on the tooth surface or in the gingival crevice when P. gingivalis is present.     

Purification and characterization of surface fibrils from taxonomically typical Actinomyces viscosus WVU627.

Fibrils of Actinomyces viscosus WVU627 (numerical taxonomy cluster 1) were obtained by homogenization and purified by ultrafiltration, ammonium sulfate precipitations, gel filtration, and ion-exchange chromatography. Electron microscopy and resolution of a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis attested to the purity of the preparation. Purified fibrils were composed mainly of protein; small quantities of carbohydrate and phosphorus were detected. Immunoelectrophoresis revealed only a single precipitable antigen, which migrated slightly toward the anode, in reactions between purified fibrils and antiserum raised against either whole bacterial cells or the purified fibrils themselves. Immunoelectron microscopy with ferritin-conjugated antifibril antibody hemagglutination inhibition, and bacterial agglutination tests demonstrated that fibrils of Actinomyces viscosus cluster 1 strains shared some common antigens with clusters 2, 3, 4 and 6, but did not cross-react with typical Actinomyces naeslundii of cluster 5. Stability tests revealed that after heat or alkali treatment, the fibrils lost their antigenicity and disappeared from electron micrographs. They were affected less by sodium dodecyl sulfate, sonic, or acid treatments.     

Adhesion and surface-aggregation of Candida albicans from saliva on acrylic surfaces with adhering bacteria as studied in a parallel plate flow chamber

Adhesive interactions between Candida albicans and oral bacteria are generally thought to play a crucial role in the microbial colonization of denture acrylic, which may lead to denture stomatitis. This study investigated the influence of saliva on the adhesive interactions between C. albicans and Streptococcus sanguis or Actinomyces naeslundii on denture acrylic. First, bacteria were allowed to adhere to the acrylic surface from a flowing suspension, and subsequently yeasts were flowed over the acrylic surface. The organisms were assayed in the presence or absence of human whole saliva. All experiments were carried out in a parallel plate flow chamber and enumeration was done in situ with an image analysis system. In the absence of adhering bacteria, adhesion of C. albicans from buffer was more extensive than from saliva. However, in the presence of adhering bacteria, yeast adhesion from saliva was increased with respect to adhesion of yeasts from buffer, indicating that specific salivary components constitute a bridge between bacteria and yeasts. In all cases, yeast aggregates consisting of 3 to 5 yeast cells were observed adhering to the surface. A surface physico-chemical analysis of the microbial cell surfaces prior to and after bathing the microorganisms in saliva, suggests that this bridging is mediated by acid-base interactions since all strains show a major increase in electron-donating surface free energy parameters upon bathing in saliva, with no change in their zeta potentials. The surface physico-chemical analysis furthermore suggests that S. sanguis and A. naeslundii may use a different mechanism for adhesive interactions with C. albicans in saliva.     

Novobiocin-resistant mutants of Streptococcus sanguis with reduced cell hydrophobicity and defective in coaggregation

Mutants of Streptococcus sanguis resistant to novobiocin (NovR-mutants) were isolated after mutagenesis of strain Challis with ethyl methanesulphonate. The resistance phenotype was transferred by DNA-mediated transformation back into the parent strain at high frequency suggesting resistance was due to mutation(s) in a single gene or in closely-linked genes. Cells of NovR-mutants had normal morphology and secreted similar proteins to the wild-type strain. However, mutant cultures had slower growth rates, the mutant cells had reduced hydrophobicity, and they showed a reduced degree of coaggregation with Actinomyces viscosus and Actinomyces naeslundii. Cell envelopes prepared from NovR-mutants differed from wild-type cell envelopes in that they (a) were impaired in ability to coaggregate with A. viscosus cells, and (b) had altered protein composition as detected by SDS-PAGE. The results suggest that hydrophobic proteins in the cell envelope of S. sanguis may be necessary for coaggregation of this bacterium with actinomycetes.     

Effect of selected bacteria on mycelial transformation of cells of Candida albicans

Influence of selected bacteria representing typical physiological flora of mucous membranes of man on transformation of Candida albicans from yeast phase to mycelial phase was evaluated, E. coli, S. viridans and S. faecalis inhibited to different degree mycelial transformation of fungal cells. A degree of inhibition in the case of E. coli was proportional to the period of preliminary culture of strains in medium containing serum while streptococci inhibited mycelial transformation mainly after 4 and 24 hr of preliminary culture. Production of factor(s) inhibiting mycelial transformation of C. albicans by E. coli was induced by direct contact with fungal cells and by low molecular weight substances produced by C. albicans. Streptococci produced inhibiting factors even when fungal cells or their metabolites were absent in the medium.     

In Vitro Inhibition of Oral Candida albicans by Chicken Egg Yolk Antibody (IgY)

This study was conducted to measure Candida albicans-specific chicken egg yolk antibody (IgY) inhibition of fluconazole-sensitive and resistant strains of C. albicans in order to assess potential use in the prevention and treatment of oral candidiasis. In this study, laying hens were immunized, and IgY was extracted by water dilution. The Minimal Inhibitory Concentrations (MICs) of IgY for inhibiting C. albicans growth were determined using the broth microdilution method from the CLSI M27-A2 protocol. Fluconazole (FLC) was used as the control. The results were analyzed with the chi(2) test. The anti-Candida titer of anti-C. albicans IgY was 1:12,000. The concentration of the IgY extract that effectively inhibited the growth of C. albicans was between 1.25 g/l and 5.0 g/l, and the efficacy rate was 82.98% during the observed 24-48 h time period. No correlation was recorded between the drug resistance of FLC and growth inhibition by IgY. It was concluded that anti-C. albicans IgY inhibited the growth of C. albicans in vitro and there was no correlation between the drug resistance of FLC and the growth inhibition by IgY (P > 0.99).     

Inducible defense mechanism against nitric oxide in Candida albicans

The yeast Candida albicans is an opportunistic pathogen that threatens patients with compromised immune systems. Immune cell defenses against C. albicans are complex but typically involve the production of reactive oxygen species and nitrogen radicals such as nitric oxide (NO) that damage the yeast or inhibit its growth. Whether Candida defends itself against NO and the molecules responsible for this defense have yet to be determined. The defense against NO in various bacteria and the yeast Saccharomyces cerevisiae involves an NO-scavenging flavohemoglobin. The C. albicans genome contains three genes encoding flavohemoglobin-related proteins, CaYHB1, CaYHB4, and CaYHB5. To assess their roles in NO metabolism, we constructed strains lacking each of these genes and demonstrated that just one, CaYHB1, is responsible for NO consumption and detoxification. In C. albicans, NO metabolic activity and CaYHB1 mRNA levels are rapidly induced by NO and NO-generating agents. Loss of CaYHB1 increases the sensitivity of C. albicans to NO-mediated growth inhibition. In mice, infections with Candida strains lacking CaYHB1 still resulted in lethality, but virulence was decreased compared to that in wild-type strains. Thus, C. albicans possesses a rapid, specific, and highly inducible NO defense mechanism involving one of three putative flavohemoglobin genes.     

Kidney homogenate, a test medium to determine the inhibitory effect of antimycotics on yeasts.

The test procedure described in this paper aims at assessing the effectiveness of antimycotics against yeasts. The inhibitory effect on Candida albicans growth is determined in the presence of kidney homogenate, an in vitro test medium offering fungal growth conditions similar to physiological conditions. The inoculum consists of kidney material from Candida-infected mice. By dilution with kidney material from uninfected mice the initial number of viable particles is standardised. Specimens withdrawn from this inoculum are mixed with different concentrations of the test substance and incubated for 24 hours. The inhibition of fungal growth is assessed on the basis of the number of colony-forming units in comparison with controls to which no test substance was added. The growth phase of C. albicans during incubation in vitro resembles that observed in the kidney in vivo, i.e. besides yeast cells, the fungus produces a great number of pseudomycelia and mycelia. The results of the tests conducted with the top-ranking antimycotics give good evidence of their therapeutic efficacy. The testing of further active substances revealed advantages of the new test model over existing test methods.     

Antifungal activity of the amyrin derivatives and in vitro inhibition of Candida albicans adhesion to human epithelial cells

AIMS: The antifungal activity of amyrin pentacyclic triterpene and 15 synthetic derivatives was evaluated against Candida species. Additionally, inhibition of adhesion of Candida albicans to human epithelial cells in vitro was determined. METHODS AND RESULTS: Esterification of alpha- and beta-amyrin with a variety of acyl chlorides produced a series of analogue derivatives. These substances were synthesized to evaluate the antifungal properties against Candida species. Among the 15 derivatives, alpha- and beta-amyrin formiate (2) and alpha- and beta-amyrin acetate (3) were the most active, inhibiting all the Candida species tested in concentrations that ranged from 30 to 250 microg ml(-1). alpha- and beta-amyrin formiate inhibited the adhesion ability of C. albicans to buccal epithelial cells (BEC) in 65.3%. CONCLUSIONS: alpha- and beta-amyrin formiate and alpha- and beta-amyrin acetate derivatives exhibited potential antifungal activity against Candida spp. and amyrin formiate showed inhibition of the adhesion ability of C. albicans to buccal epithelial cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that two derivatives of amyrin pentacyclic triterpene exhibited significant antifungal activity against Candida species. Additionally, alpha- and beta-amyrin formiate was as effective as fluconazole in inhibiting the adhesion of C. albicans to buccal epithelial cells.     

Numerical taxonomy and laboratory identification of Actinomyces and Arachnia and some related bacteria.

A numerical taxonomic study was made on 49 facultative anaerobic Gram-positive filamentous and/or diphtheroidal organisms isolated from dental plaques, carious dentin and faeces, together with 63 reference strains belonging to the genera Actinomyces, Arachnia, Bifidobacterium, Actinobacterium, Propionibacterium, Eubacterium and Lactobacillus. They were examined for 90 unit characters covering a wide range of tests and properties. The data were subjected to computer analysis in which the simple matching coefficient (SSM) and the similarity index (SJ) were calculated, and the results of single linkage techniques and an unweighted average linkage cluster analysis technique were compared. The strains fell into six major groups (phena). The Actinomyces strains were recovered in two phena; the first contained Actinomyces israelii and the other facultative anaerobic Actinomyces, including subclusters equal to taxospecies of A. odontolyticus and A. viscosus/A. naeslundii, while the other phenon corresponded to the genera Arachnia, Actinobacterium, Bifidobacterium and Propionibacterium. The groups of Arachnia and Actinobacterium each contained one species, representing taxospecies of Arachnia propionica and Actinobacterium meyerii. Taxonomic criteria, both constant and discriminative, were selected to form a diagnostic table useful for laboratory identification of this group of organisms. Immunofluorescence supported the numerical data.     

Effects of compounds found in Nidus Vespae on the growth and cariogenic virulence factors of Streptococcus mutans

Nidus Vespae (honeycomb) is a kind of traditional Chinese medicine that has been demonstrated to inhibit the growth and acid-production of oral cariogenic bacteria. Subsequent studies showed that the chloroform/methanol (Chl/MeOH) chemical extraction of Nidus Vespae was the most effective inhibitor of growth and acidogenicity of Streptococcus mutans. In this study, we isolated the chemical compounds of the Nidus Vespae Chl/MeOH extraction, tested their antimicrobial activity against six cariogenic bacteria and further evaluated the acid inhibition properties, anti-F-ATPase activity and anti-LDH activity against S. mutans. The isolated flavonoids, quercetin and kaempferol, inhibited the growth of bacteria (S. mutans, Streptococcus sobrinus, Streptococcus sanguis, Actinomyces viscosus, Actinomyces naeslundii and Lactobacillus rhamnosus) with minimum inhibitory concentrations (MICs) ranging from 1 to 4 mg/ml and minimum bactericidal concentrations (MBCs) from 4 to 16 mg/ml. In addition, quercetin and kaempferol at sub-MIC levels significantly inhibited acidogenicity and acidurity of S. mutans cells. Treated with the test agents, the F-ATPase activity was reduced by 47.37% with 1mg/ml quercetin and by 49.66% with 0.5mg/ml kaempferol. The results showed that quercetin and kaempferol contained in Chl/MeOH extraction presented remarkably biological activity, suggesting that Nidus Vespae might be useful as a potential preventive and therapeutic agent in dental caries.     

Surface localization of sialic acid on Actinomyces viscosus

This study reports the presence of sialic acid in Actinomyces viscosus strains T14V and T14AV. Mild acid hydrolysis of whole organisms released a compound which reacted positively in the periodate-thiobarbituric acid, direct Ehrlich's and resorcinol assays, and which co-chromatographed on paper with authentic N-acetylneuraminic acid. Strain T14V contained 10-fold greater concentrations of sialic acid than did strain T14AV. Sialic acid content was dependent upon the stage of growth of the culture, reaching a maximum in early stationary phase. Epifluorescence microscopy of fluorescein isothiocyanate (FITC)-conjugated Limulus polyphemus agglutinin (LPA), a lectin specific for sialic acid, revealed a uniform distribution of bound lectin on the surfaces of strains T14V and T14AV. Additional evidence for surface localization was obtained by demonstration of whole-cell agglutination of both strains with LPA. All LPA interactions with A. viscosus were inhibited by the presence of 0.1 M-N-acetylneuraminic acid. Neuraminidases from Clostridium perfringens, Arthrobacter ureafaciens and Vibrio cholerae did not release detectable amounts of sialic acid, but the extracellular enzyme from A. viscosus cleaved amounts equivalent to those obtained by acid hydrolysis. Other laboratory strains (W1053, M100, W859, 5-5S, RC45, ATCC 19246, and 'binder') as well as recent clinical isolates of A. viscosus were agglutinated by LPA and released sialic acid upon mild acid hydrolysis. Surface-available sialic acid has been implicated in the inhibition of alternative complement pathway activation and subsequent opsonophagocytosis. Thus the occurrence of surface sialic acid in A. viscosus may represent a mechanism of pathogenesis for this oral bacterium.