Distribution of antigenic determinants between Actinomyces viscosus and Actinomyces naeslundii.

A total of 18 monoclonal antibodies was raised against whole cells of Actinomyces viscosus and Actinomyces naeslundii. The monoclonal antibodies were used to determine the cross-reacting patterns among 26 strains of these species. Eleven different antigenic determinants were found. The specificity profiles of the antibodies indicated that the antigenic determinants of A. viscosus and A. naeslundii were arranged in a complicated mosaic. Extensive cross-reactions occurred between A. viscosus strains and strains of "typical" and "atypical" A. naeslundii. However, cross-reactions were rare between the two groups of A. naeslundii. A. viscosus appears to occupy a "middle position" between the two A. naeslundii groups. In addition to their value in seroclassification, some of the monoclonal antibodies were found to be useful in the identification of these species. One monoclonal antibody appeared to be selective for the "typical" A. naeslundii group. A. viscosus and "atypical" A. naeslundii-specific antibodies were also found, though they did not label every strain in their respective clusters. A. viscosus detection might be improved if mixtures of monoclonal antibodies were used.

Longitudinal microbiological investigation of a hospitalized population of older adults with a high root surface caries risk.

Intact root surfaces of chronically hospitalized subjects were sampled periodically to enumerate bacterial species believed to be associated with root caries. Bacteria were cultivated and enumerated using a series of selective and enriched media. Microbial counts, isolation frequencies, and percent cultivable flora data were analyzed for caries-active and caries-free surfaces and subjects. S. mutans, S. sanguis, A. viscosus, A. naeslundii, total lactobacilli, and Veillonella accounted for a mean of less than 20% of the cultivated flora, with mitis salivarius agar cultivable streptococci averaging less than 5%. The microbial count data were highly variable, precluding the finding of significant differences in caries association for either subjects or sites. Streptococci, especially S. mutans, correlated highly with lactobacilli in the samples.

Streptococcus mutans and Lactobacillus detection in the assessment of dental root surface caries risk.

Forty-five subjects contributing 150 root surfaces with gingival recession were sampled seven times over a 32-month period. We calculated isolation frequencies of lactobacilli on selective Rogosa SL agar and S. mutans in a sensitive enrichment broth and on mitis salivarius agar. Both S. mutans and lactobacilli were isolated more frequently from surfaces which became carious than from those remaining caries-free. Isolation frequencies were also higher for caries-free surfaces in caries-active subjects than for caries-free surfaces in caries-inactive subjects. The presence or absence of S. mutans and lactobacilli in samples taken at baseline could discriminate between subjects who were to be root-caries-active and those who were to remain root-caries-inactive during the subsequent observation period. Moreover, if both bacteria were detected or only S. mutans was detected on a root surface at its entry into the study, that surface had a greater risk for developing a root lesion. However, the tests could not predict which root surfaces within the mouths of caries-active subjects were to become carious. Analysis of the data suggests that simple microbiological detection tests may be useful in identifying patients at high risk of root caries.

Identification of bacteria in immunopathologic mechanisms of human dental pulp.

Serial frozen sections from human dental pulp were used for the identification of oral bacteria in immunopathologic mechanisms. Sera raised against Actinomyces viscosus, A. naeslundii, Bacteroides gingivalis, and B. melaninogenicus ss. intermedius, commercial sera against human immunoglobulins, complement, and monoclonal antibodies against human T cells were used in a double-staining immunofluorescence technique. Sections of dental pulp from normal teeth showed no penetration of bacteria or bacterial antigens and no signs of inflammation. A unique aspect of the present study was the demonstration that penetrating bacteria and bacterial antigens in the pulp of involved teeth were always associated with antibodies and frequently also with complement. A. viscosus has been found most frequently in complement-fixing immune complexes followed by B. gingivalis. A. naeslundii and B. melaninogenicus ss. intermedius were found only in complexes with antibodies. The involvement of plasma cells and T cells was also demonstrated. In the dental pulps of diseased teeth, cytotoxic and Arthus type immunopathologic reactions occurred.

Chemical and immunological comparison of surface fibrils of strains representing six taxonomic groups of Actinomyces viscosus and Actinomyces naeslundii.

Human isolates of Actinomyces viscosus and Actinomyces naeslundii have been divided into six clusters in a numerical taxonomy study. Surface fibrils of strains representing these clusters were isolated and purified. Chemical analyses revealed that the major component of all fibrils was protein and that although differences in percentages of specific amino acid residues were found, the relative proportions of basic, acidic, polar uncharged, and nonpolar amino acids were rather similar among clusters. All of the fibrils except those from strain B236 (cluster 2) either failed to migrate or penetrated only slightly into gels during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even after boiling, reduction, or alkylation. Immunological studies by electron microscopic examination of fibril-antibody immunocomplexes, whole bacterial cell agglutination, inhibition of hemagglutination, and immunofluorescence by using antifibril antisera and antibodies demonstrated that strains of typical A. naeslundii (cluster 5) have a specific fibril-associated antigen(s) distinct from those of strains of other clusters. Cross-reactions for atypical A. naeslundii (cluster 3) were few. The fibrils from A. viscosus clusters 1, 2, 4, and 6 demonstrated several cross-reactions. By absorbing antifibril antibodies with cross-reactive strains it was possible to obtain cluster-specific antibodies, as determined by whole cell agglutination, only for cluster 5. Absorbed antifibril antisera for both A. naeslundii clusters 3 and 5 were specific by indirect immunofluorescence, whereas anti-cluster 1 fibril antisera cross-reacted only with other A. viscosus cluster representatives. Purification of Actinomyces fibrils by methods used for appendages of other species yields preparations containing common antigens among taxonomic groups. However, absorbing antifibril antisera, gamma globulin, or both has promise for producing cluster-specific reagents useful in identification.

Prevalence of root surface caries among institutionalized older persons.

Fifty-nine residents of a chronic hospital (average age 67.9 years) were examined visually for root surface caries. Root lesions were found to be present in 44 of the residents and were located most frequently on the proximal surfaces of anterior teeth. The number of coronal DF surfaces, age and number of retained teeth were the factors found to be helpful in discriminating between persons with and without root surface caries.

Comparison of selective broth and plating methods for isolation of Streptococcus mutans from root surface dental plaques.

Approximately 150 sound root surfaces in 44 subjects prone to root surface caries were sampled longitudinally to determine the microbial flora associated with root caries initiation. During the first 16 months of the study, a comparison of Streptococcus mutans recovery was made by using three bacteriological media: mitis-salivarius agar (MSA), mitis-salivarius-bacitracin-sucrose agar (MSB), and a partially selective mannitol-containing broth. Total streptococcal and S. mutans populations were found to be much lower than in previous reports. MSB was more selective; S. mutans was detected in many samples when its numbers were too low for isolation on MSA. However, recovery of S. mutans was greater on MSA than on MSB for most samples yielding colonies on both media. Mannitol-containing broth used as an enrichment medium yielded the highest frequency of S. mutans isolation among the three media.

Sialidase-enhanced lectin-like mechanism for Actinomyces viscosus and Actinomyces naeslundii hemagglutination.

Laboratory strains representing six numerical taxonomy clusters and fresh isolates of human Actinomyces viscosus and Actinomyces naeslundii were studied by standard flocculation slide tests for the ability to hemagglutinate erythrocytes (RBC) from various animal species. Human AB and horse RBC were agglutinated more frequently and rapidly than others; guinea pig RBC were agglutinated by only a few strains. Human AB RBC were selected for studies of hemagglutination mechanisms. Treatment of RBC with clostridial neuraminidase (NTRBC) greatly enhanced hemagglutination for almost all strains. In hapten inhibition experiments in which various concentrations of sugars were used, beta-galactosides were the most effective inhibitors of hemagglutination for both RBC and NTRBC; inhibition of NTRBC agglutination required higher concentrations. Soybean lectin agglutinated both RBC and NTRBC but not Actinomyces cells. NTRBC agglutinated at a 125-fold-lower concentration. Hemagglutination was sensitive to ethylenediaminetetraacetate for one strain tested. Hemagglutination reactions were reversible by addition of beta-galactosides. The ability of Actinomyces strains to "prime" RBC for hemagglutination by removing sialic acid to expose more penultimate beta-galactoside sites was studied by recycling Actinomyces-agglutinated RBC which were dispersed with a lactose solution and washed free of bacteria (primed RBC). Priming in this manner augmented subsequent hemagglutination by indicator Actinomyces strains and made the RBC more sensitive to agglutination by soybean lectin. The priming ability of Actinomyces strains generally correlated with the amount of sialic acid removed from primed RBC. Strains representing the numerical taxonomy clusters differed in both their hemagglutinating and priming activities. Cluster 5 strains (typical A. naeslundii) were good agglutinators of RBC, NTRBC, and primed RBC but were poor primers. Cluster 3 strains (atypical A. naeslundii) were the weakest hemagglutinators but could prime RBC adequately for subsequent agglutination by other strains. Together, these data indicate that Actinomyces hemagglutination proceeds via a two-step mechanism: (i) neuraminidase removal of terminal sialic acid and (ii) lectin-like binding to exposed beta-galactoside-associated sites on the RBC. Strains differ in the extent to which they can perform the two functions, and this specificity may relate to their taxonomic classification.

Mannose-contaminating agglutinin for Actinomyces viscosus and Actinomyces naeslundii.

Rapid agglutination of Actinomyces viscosus and Actinomyces naeslundii cells by D-mannose solutions was observed during studies of their attachment to mammalian cells in vitro. The specificity of the agglutination reaction was studied by slide agglutination tests and by measuring the rate of decrease in optical density of bacterial phosphate buffer suspensions caused by the setting of bacterial aggregates. Actinomyces cells were agglutinated by protein-containing mannose solutions of several chemical suppliers. Solutions of sugars other than D-mannose and solutions of mannitol and mannan all failed to agglutinate A. viscsus and A. naeslundii. "Mannose-enhanced" agglutination was impaired by boiling or autoclaving the mannose but was not affected by heating the bacteria, the presence of chloramphenicol, running the assay in the cold, or incorporating any of several commercially purchased sugars in the reaction mixture. During these hapten inhibition experiments, only 6-deoxy-L-talcose-containing extracts of an A. viscosus strain retarded the rate of mannose-enhanced agglutination. Protein-containing fractions of D-mannose mother liquors also agglutinated cells of A. viscosus and A. naeslundii. Other species of oral gram-positive rods were not agglutinated by mannose solutions. Together the data indicate that plant seed-derived D-mannose contains a protein-associated agglutinin for A. viscosus and A. naeslundii which may function via a "lectin-like" selective affinity for the unique cell wall sugar 6-deoxy-L-talose.

Wall carbohydrate antigens of A. israelii.

1) A. israelii strains carry common carbohydrate determinants. 2) Type strains NCTC 4860, ATCC 10048 and ATCC 12102 only carry the common determinants. 3) More recent isolates of A. israelii/1 can carry additional antigenic determinants in the cell wall. 4) These additional antigens can be separated by chromatography on Sephadex G-200.

A longitudinal epidemiological study on dental plaque and the development of dental caries--interim results after two years.

1. During the two-year period, caries developed at 20% of the target premolar sites. The attack rate for these surfaces was similar in the plaque panel and the other subjects in the study. 2. The microbial composition of plaque samples from caries-free sites and from carious sites before and after radiographic detection of lesions was broadly similar. 3. Numerical domination of particular sites by S mutans before detection of caries can occur, but has only been observed so far in 2 of 15 sites. 4. Pooled date from sites which have developed lesions indicate a rise in the isolation frequency and mean numbers of S. mutans after detection of caries. This trend was particularly obvious in the one subject who developed bilateral lesions by the second examination and in three of four sites where caries was detected at the fourth examination. Similar observations have been made with lactobacilli. 5. In two of 15 instances no isolations of S mutans were made from sites which developed caries. 6. To date, no single species appears to be uniquely associated with the onset of dental caries.

Antigens from Actinomyces species and their value in identification.

Actinomyces have a complex antigenic structure that includes neutral cell wall carbohydrate and some polypeptide containing charged antigens. Wall carbohydrate can be prepared in a relatively pure form from cell walls or by DEAE Sephadex chromatography of acid extracts and supernatant culture fluid. Charged antigens are generally eluted in groups by batch-elution from columns. Cell wall carbohydrate can be responsible for species-specific reactions and cross-reactions, and strains may possess more than one cell wall carbohydrate determinant. The charged antigens also can be species specific, but they cause some cross-reactions, particularly those between the A israelii serotypes. Autoclave extracts of strains, tested against antiserums reacting with cell wall carbohydrate, may be valuable in routine identification of isolates.