Cross-kingdom interaction of Candida albicans and Actinomyces viscosus elevated cariogenic virulence.

OBJECTIVE: How the interactions between Candida albicans and Actinomyces viscosus contributed to the root caries was not clear. This study aimed to investigate their cross-kingdom interactions on the biomass and the cariogenic virulence in dual-species biofilms. DESIGN: Suspensions of C. albicans and A. viscosus were formed the mono and polymicrobial biofilms in vitro. Crystal violet assay, viable plate count, scanning electron microscopy and fluorescence in situ hybridization were used to analyze the biomass and biofilm structure. Glycolytic pH drop and the spectrophotometric method were used to evaluate the acid production and hydroxyapatite dissolution, respectively. The exopolysaccharide production was measured by the anthrone-sulfuric acid method, while the adhesion force was measured by atomic force microscopy. RESULTS: The biomass and colony-forming units of mixed-species were significantly increased compared to that of the mono-species at 24 h, 48 h, 72 h. The structure of dual-species biofilm had more microcolonies and was much denser. The dual-species biofilms significantly decreased the pH value and damaged the hydroxyapatite compared with the mono-species biofilms at various time points, indicating the strong cariogenic virulence. Moreover, the dual-species biofilms significantly enhanced the exopolysaccharide production and adhesion force suggesting the increase of biofilm adhesion. CONCLUSIONS: Cross-kingdom interactions of C. albicans and A. viscosus significantly elevated the biomass and cariogenic virulence of dual-species biofilm.

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.

Different type 1 fimbrial genes and tropisms of commensal and potentially pathogenic Actinomyces spp. with different salivary acidic proline-rich protein and statherin ligand specificities.

Actinomyces spp. exhibit type 1 fimbria-mediated adhesion to salivary acidic proline-rich proteins (PRPs) and statherin ligands. Actinomyces spp. with different animal and tissue origins belong to three major adhesion types as relates to ligand specificity and type 1 fimbria genes. (i) In preferential acidic-PRP binding, strains of Actinomyces naeslundii genospecies 1 and 2 from human and monkey mouths displayed at least three ligand specificities characterized by preferential acidic-PRP binding. Slot blot DNA hybridization showed seven highly conserved type 1 fimbria genes (orf1- to -6 and fimP) in genospecies 1 and 2 strains, except that orf5 and orf3 were divergent in genospecies 1. (ii) In preferential statherin binding, oral Actinomyces viscosus strains of rat and hamster origin (and strain 19246 from a human case of actinomycosis) bound statherin preferentially. DNA hybridization and characterization of the type 1 fimbria genes from strain 19246 revealed a homologous gene cluster of four open reading frames (orfA to -C and fimP). Bioinformatics suggested sortase (orfB, orf4, and part of orf5), prepilin peptidase (orfC and orf6), fimbria subunit (fimP), and usher- and autotransporter-like (orfA and orf1 to -3) functions. Those gene regions corresponding to orf3 and orf5 were divergent, those corresponding to orf2, orf1, and fimP were moderately conserved, and those corresponding to orf4 and orf6 were highly conserved. Restriction fragment length polymorphism analyses using a fimP probe separated human and monkey and rat and hamster strains into phylogenetically different groups. (iii) In statherin-specific binding, strains of A. naeslundii genospecies 1 from septic and other human infections displayed a low-avidity binding to statherin. Only the orf4 and orf6 gene regions were highly conserved. Finally, rat saliva devoid of statherin bound bacterial strains avidly irrespective of ligand specificity, and specific antisera detected either type 1, type 2, or both types of fimbria on the investigated Actinomyces strains.

The diversity and distribution of the predominant ribotypes of Actinomyces naeslundii genospecies 1 and 2 in samples from enamel and from healthy and carious root surfaces of teeth.

The bacterial communities associated with root caries are highly diverse and undergo succession during lesion formation. Consequently, root caries is said to have a polymicrobic etiology, typified by variation in the predominant species among samples from different lesions. Despite the polymicrobic etiology, A. naeslundii genospecies 1 and 2 (previously A. viscosus) have consistently been shown to be associated with root caries in humans; they predominate in some lesions and have been suggested to play a significant role in the disease. Several genetic variants of A. naeslundii are known to be present among the oral A. naeslundii population of an individual. The current study was initiated to explore the possibility that a variant in these A. naeslundii populations had characteristics which made it best fitted to colonize or promote root-surface caries lesions. Using ribotyping to detect variants, we tested the hypothesis that 'a ribotype of A. naeslundii best fitted to the environment would be selected and predominate in the A. naeslundii population of lesions'. Samples of plaque from enamel, normal root surfaces, plaque overlying the lesion, and material from within the lesion were taken from nine patients with soft root caries. The flora from 14 lesions and 9 enamel sites was analyzed on selective and non-selective media, and A. naeslundii genospecies were identified by serology. We ribotyped 972 isolates, showing 54 different patterns. Between 6 and 20 ribotypes were isolated from eight of nine patients. In general, each site from a patient showed a similar distribution of ribotypes. These results do not support the hypothesis and suggest that any phenotypic characters that allow A. naeslundii genospecies 1 and 2 to colonize or contribute to the formation of root-caries lesions are common among strains identified by ribotyping.

Powdered milk micellar casein prevents oral colonization by Streptococcus sobrinus and dental caries in rats: a basis for the caries-protective effect of dairy products.

Three animal studies were performed to investigate the influence of the macromolecular structure of milk casein on caries incidence and the possible ecological changes of the oral microbiota by such casein fractions. Towards this end, rats were infected with mixed bacterial suspensions of Streptococcus sobrinus OMZ 176 and Actinomyces viscosus Ny1. Various milk protein fractions were incorporated into carefully balanced powdered cariogenic diets to constitute the sole major protein component. Diets containing micellar casein had a pronounced and highly significant effect on almost all clinical and microbiological parameters examined. Both the formation of advanced dentinal fissure (B) and smooth surface (E) caries lesions was inhibited by diets containing micellar casein; this caries-inhibiting effect appeared to be due mainly to modifications within the plaque microbiota. The proportion of S. sobrinus in the oral cavity of rats was reduced (73-80%) by micellar casein-containing preparations, whereas the A. viscosus population was increased. Both these microbiological parameters were always negatively correlated. This appears to be the first example of a food component other than dietary sugars, selectively modifying the composition of the dental plaque microbiota of rats in such a way as to reduce its pathogenic potential. It also demonstrates the importance of establishing a molecular basis for the role of food components, which prove to be beneficial to oral health.

Co-aggregation as a virulent factor of Streptococcus sanguis isolated from infective endocarditis.

The pathogenicity of strains of the Streptococcus sanguis group, isolated from infective endcarditis, was studied by measuring the development of subcutaneous abscesses in mice after infection with S. sanguis and Actinomyces viscosus either singly or as co-aggregated pairs. The pathogenicity of the co-aggregates was also examined in various viable combinations of the two bacterial species. More abscesses were formed by A. viscosus than the S. sanguis group including clinical isolates. Abscess formation by co-aggregates of combinations of each isolate and A. viscosus produced a higher percentage of abscess formation than those caused by infection with a pure suspension of A. viscosus or tested streptococci. Co-aggregated cells were more resistant to phagocytosis and killing by neutrophils in vivo. These results indicated that S. sanguis group streptococci isolated from infective endocarditis are able to co-aggregate and resist phagocytosis. The ability of co-aggregation of S. sanguis may serve as a survival mechanism in a host defense system and may be linked with virulence of this bacteria.

[A study on biological properties of fimbriae of A. viscosus. III. Adherence activity of fimbriae of A. viscosus].

OBJECTIVE: To investigate the adherence activity of two types of fimbriae of A. viscosus on tooth surface or with S. sanguis 34. METHODS: The inhibited adherence tests, coaggregation tests and the inhibited coaggregation tests were done. RESULTS: 1. The purified type I and type II fimbriae inhibited the adherence of A. viscosus to salivary-treated hydroxyapatite (SHA) and the two specific IgG to type I and type II fimbriae blocked the adsorption of strain T14V, strain 5519 and strain 5951 to SHA; 2. Only type II fimbria indirectly mediated the visible agglutination of S. sanguis 34 and only IgG to type II fimbriae inhibited coaggregation of strain T14V and strain 5915 with S. sanguis 34. CONCLUSION: Type I and type II fimbriae have adherence ability, and only type II fimbria has the agglutination activity. Additionally, the methods which were used to prepare fimbriae don't damage the biological activity of fimbriae.

Effects of Actinomyces amphiphile on the fluidity of endothelial cells: a spin label study.

Actinomyces amphiphile (AcA) is an amphipathic molecule produced by Actinomyces viscosus that exhibits several biological activities. The effect of AcA on the fluidity and permeability of the plasma membrane in human umbilical vein endothelial cells was analyzed by a spin label method with 5- and 16-stearic acid nitroxide labels (SAL). These labels help to visualize the fluidity at the shallow (5-SAL) and deep (16-SAL) portions of the lipid bilayer. Cells were incubated with and without AcA (control) at 37 degrees C for 6 hours, and membrane fluidity was periodically measured. Another spin label, 4-(N, N-dimethyl-N-hexadecyl) ammonium-2, 2, 6, 6-tetramethyl-piperidine-1-oxyliodine (CAT-16), was also used to assess the physical state of the cell surface. The order parameter of 5-SAL was significantly lower in the cells incubated with AcA than in control cells after the six-hour incubation. The motion parameter of 16-SAL was significantly lower in AcA-treated cells than in controls after 4 and 6 hours of incubation. These findings indicated that the AcA increased the fluidity. There were no significant differences between the AcA-treated and control cells incubated for only 2 hours. In addition, there were no differences in CAT-16 measurements between AcA-treated and control cells. The release of endoplasmic lactate dehydrogenase (LDH) into the medium tended to increase in the AcA-treated vs. the control cells. LDH release increased in both a dose- and time-dependent manner, indicating that AcA increased the permeability of plasma membranes. These findings suggest that AcA alters the biophysical properties of the plasma membranes of endothelial cells, affecting membrane function.

Expression of the LFA-1 beta molecule on peripheral blood leukocytes of patients with early-onset periodontitis: effects of dental plaque microbes.

The effect of dental plaque bacteria on LFA-1 beta expression on peripheral blood leukocytes was studied in 20 patients with early-onset periodontitis and in 10 healthy controls. Stimulation of PMN with selected dental plaque bacteria which play a role in the pathogeny of periodontitis significantly increased the expression of LFA-1 beta in the group of patients as compared with the controls.

Experimental root surface caries in hamsters the development of the disease after inoculations of two types of cariogenic bacteria.

The aim of this study was to investigate the development of root surface caries in hamsters fed a high-sucrose diet over a 24-week period after inoculations of two types of cariogenic bacteria. Twenty-one day old male golden hamsters (n = 103) were divided into 5 groups. Four groups were given diet 2000, and one group was given a stock diet CE-2, Of the groups given diet 2000, three groups were infected with Actinomyces viscosus ATCC 15987 and Streptococcus mutans NTCC 10449 separately (AV and SM groups) or in combination (AVSM group), and one group remained uninfected. A grid method was used to evaluate the plaque accumulation, alveolar bone loss, and root surface caries. After 12 weeks, root surface caries developed mainly on the first mandibular molars in the three infected groups. At 24 weeks, the prevalence of root surface caries was highest in the AV group, but root caries scores were not significantly different among the three infected groups. In the groups SM and AVSM, the molar crowns were extensively destroyed by caries, while in the AV group the crowns were almost intact. It was concluded that challenge with Actinomyces viscosus may be appropriate to study root surface caries in hamsters.

Microbial induction of dentinal caries in human teeth in vitro.

The ability of Lactobacillus casei, Streptococcus sobrinus, Actinomyces viscosus, and Streptococcus salivarius to induce dental caries was determined in vitro. A class I cavity (depth: 2mm) was prepared in extracted human caries-free premolars to make dentin blocks. The blocks were inoculated with these four bacterial strains in a monoinfective fashion and were incubated under anaerobic conditions. In addition to the monoinfection groups, mixed-infection groups of L. Casei with S. sobrinus or A. viscosus were also prepared. Half of the culture medium was renewed every 3 days, and the pH of the medium was measured. After 4 or 12 wk, these dentin blocks were prepared by Brown-Brenn staining and by contact microradiography for light microscopic observation and for immunohistochemical staining. The final pH of the S. salivarius group was the highest among the experimental groups, at approximately 5.1; that of the others was approximately 4.3. Bacterial invasion into the dentinal tubules was observed in all but the S. salivarius group. Among the monoinfection groups, the S. sobrinus group showed the highest invasion rate, followed by the A. Viscosus group and the L. casei group. The invasion rate was also high in the mixed-infection groups. Immunohistochemical staining revealed invasion only by L. casei, and not by S. sobrinus and A. viscosus. The invasion rate by L. casei was higher in the mixed-infection group with either S. sobrinus or A. viscosus than in the monoinfection groups. These findings suggest that lactobacillus might play an important role in the initiation and progress of dentinal caries, and that this bacterial species might exhibit a cooperative cariogenicity when it coexists with other bacterial species that surpasses its individual cariogenicity.

In vitro demineralization by strains of Actinomyces viscosus and Streptococcus sobrinus of sound and demineralized root surfaces.

Root sections were inoculated with one of two strains of Actinomyces viscosus or a strain of Streptococcus sobrinus and then incubated for 9 days in Trypticase soy broth (TSB) containing 0.25% glucose or TSB supplemented with 0.25% sucrose (TSB+S). Lesion progression was measured from microradiographs. One section from each group was examined with a transmission electron microscope. Lesion progression was associated with a fall in the pH of the medium. However, lesion progression was not correlated with the mean 48-hour pH of the medium. In both TSB and TSB + S, lesion progression with A. viscosus was significantly greater than in the S. sobrinus group. However, the mean 48-hour pH value in TSB + S was lower than that in the S. sobrinus group. Plaque formation in TSB was, subjectively, least in the S. sobrinus group. Examination of transmission electron micrographs revealed bacteria penetrating the surfaces of the sections and extending into the lesion in the A. viscosus groups but not in the S. sobrinus group. This in vitro bacterial plaque model of root caries may be suitable for investigations of the bacterial etiology of root-surface caries and the virulence factors associated with pathogenicity.

[Studies on immunobiological activities of periodontopathic bacteria. Comparison of the activities of soluble components from Bacteroides gingivalis and Actinomyces viscosus].

It has been proposed that Bacteroides gingivalis and Actinomyces viscosus are most important agents to pathogenesis of the periodontitis and gingivitis. In this study, the influences of sonic extracts prepared from B. gingivalis and A. viscosus for DNA synthesis of murine lymphocytes, production of prostaglandin E2 (PGE2), collagenase and interleukin-1 (IL-1) from human peripheral monocyte were investigated. Furthermore, PGE2 and collagenase production by fibroblasts from human periodontal ligament (HPLF) and gingiva (Gin 1) stimulated with macrophage conditioned medium (MCM) cultured with bacterial sonic extracts were examined. The results obtained were as follows. 1. The sonic extracts (10 micrograms/ml protein) from B. gingivalis and A. viscosus showd low mitogenic activity to spleen cells, however, induced polyclonal B cell activation. 2. Although, the effect of these sonic extracts on the production of PGE2 and collagenase by human peripheral monocyte was not found, the induction of IL-1 production was recognized. 3. The culture supernatants of C3H/HeN mouse peritoneal macrophage stimulated with sonic extracts of B. gingivalis and A. viscosus were induced PGE2 and collagenase production by HPLF and Gin 1. These results suggest that the cellular components of B. gingivalis and A. viscosus may play the pathogenic roles in periodontal tissue destruction through the stimulation of macrophage and/or lymphocyte.

The effect of desalivation on coronal and root surface caries in rats.

Although the presence of coronal caries is declining in much of the Western Hemisphere, the prevalence of root surface caries is likely to increase as teeth are retained longer than heretofore. At the same time, an increasing number of the population suffer from dry mouth as a result of taking prescription drugs, with an apparent concomitant increased susceptibility to root surface caries. This study attempted to develop an animal model which would aid in the exploration of the effects of desalivation and the development of root surface caries. Animals were desalivated, infected with Actinomyces viscosus and Streptococcus mutans (sobrinus) 6715, and fed a cariogenic diet. Coronal caries developed rapidly in the animals; sufficient disease was present after two weeks to permit evaluation of potential therapeutic agents. Alveolar bone loss and root surface lesions developed in three to four weeks. S. mutans (sobrinus) and A. viscosus established readily in all animals; however, as the investigation progressed, populations of the latter declined, possibly because of the highly acidogenic environment. This model will facilitate investigation of the influence of hyposalivation and help in the exploration of agents to alleviate the adverse effects of salivary gland dysfunction.