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Polysaccharide(s) have been isolated from supernatants of broth cultures of Actinomyces viscosus, strain T6. The organism produces significant quantities (2 mg/mg of cell [dry weight], 108 mg/liter) of this polysaccharide in the absence of sucrose. Chemical analysis indicated that N-acetylglucosamine (62%) was the major component; galactose (7%), glucose (4%), uronic acid (3%), and smaller amounts of glycerol, rhamnose, arabinose, and xylose were also present. The polysaccharide(s) is only slightly soluble in aqueous solutions. Although further purification of the polysaccharides will be necessary before definitive chemical and structural studies, the formation of extracellular polysaccharides is significant because it offers a possible explanation for plaque formation by these organisms.

Germfree rats fed a high-sucrose diet were inoculated with Actinomyces viscosusstrain T14-Vi (virulent) or T14-Av (avirulent). The mean recovery of strain T14-Vi from six extracted finely ground molars of rats sacrificed after 90 days was 1.1 × 108colony-forming units (CFU). The mean recovery of strain T14-Av was 5.7 × 107CFU, which was significantly less. Strain T14-Vi caused severe alveolar bone loss, but only minimal bone loss occurred in rats infected with strain T14-Av. Scanning electron microscopy of teeth of germfree rats revealed that strain T14-Vi colonized in the fissures as well as on tooth surface areas near the gingiva; strain T14-Av also colonized in fissures but was unable to colonize the teeth near the gingiva. In studies with conventional rats fed a high-sucrose diet, streptomycin-resistant strain T14-Vi colonized on the teeth of all rats inoculated with in the order of 108or 107CFU and on the teeth of about half of the rats inoculated with 106or 105CFU. In contrast, streptomycin-resistant strain T14-Av could not be detected on the teeth of any of the rats in groups similarly inoculated. In vitro “resting” cells of both strains suspended in conventional or germfree rat saliva survived to comparable degrees. [3H]thymidine-labeled T14-Vi cells adhered well to hydroxyapatite (HA) beads and to HA beads pretreated with saliva obtained from germfree or conventional rats. In contrast, T14-Av cells adhered less well than did T14-Vi cells to HA, whereas their adherence to saliva-coated HA was negligible. Transmission electron microscopy of negatively stained T14-Vi and T14-Av cells repeatedly passed in 1% phosphotungstic acid revealed fibrils on cells of both strains. T14-Av cells were covered by large amounts of extracellular material which was presumably heteropolysaccharide; little extracellular material was present on the surface of T14-Vi cells. T14-Vi cells had a relatively low affinity for the heteropolysaccharide synthesized by strain T14-Av. Other evidence also suggested that this polysaccharide had a relatively low affinity for saliva-coated HA. Collectively, the evidence indicates that the difference in periodontopathic potential between strains T14-Vi and T14-Av results from their different abilities to colonize teeth. This difference is probably due to the lower adherence of T14-Av cells to teeth rather than to their ability to grow in the mouth. The low affinity of T14-Av cells for tooth surfaces may be due, in part, to the presence of large amounts of cell-surface-associated polysaccharide.

Both virulent (V) and avirulent (AV) strains of Actinomyces viscosusT14 are capable of colonizing the oral cavity of gnotobiotic rats, but only T14-V causes destructive periodontal disease. The basis for this difference in in vivo pathogenicity has not been adequately defined. In the present study we compared the capacities of T14-AV and T14-V to provoke in vitro extracellular release of lysosomal constituents from human polymorphonuclear leukocytes (PMNs). In serum-free cultures, viable T14-V but not T14-AV stimulated discharge of PMN lysosomes. The release response was correlated with PMN phagocytic activity; thus, PMNs readily ingested T14-V but not T14-AV. To explain these differences in PMN-bacteria interactions, subcellular fractions of T14-AV or T14-V were incubated with PMNs. A crude, insoluble sonic extract derived from T14-V caused PMN lysosome release, but a similar fraction from T14-AV was inactive. However, following extensive washing and treatment with deoxyribonuclease or sodium dodecyl sulfate, cell wall fractions of T14-AV stimulated lysosome release. These procedures apparently removed an extracellular polysaccharide slime which is synthesized by T14-AV but not by T14-V. There was a significant reduction in the capacities of viable T14-V or cell wall fractions of T14-V or T14-AV to provoke PMN lysosome release when these agents were preincubated with a slime material isolated from T14-AV. This inhibitory influence of slime was overcome by the addition of fresh or heated (56°C, 30 min) serum to the PMN-bacteria cultures. The data suggest a relationship between the abilities of the avirulent and virulent strains of A. viscosusT14 to act as periodontal pathogens in vivo and to serve as stimuli for PMN lysosome release in vitro.

A soluble polysaccharide antigen from the cell wall of Rothia dentocariosaATCC 17931 has been isolated, purified and characterized by serological and chemical procedures. The polysaccharide (RPS) was found to be located at the surface of cells grown under diverse environmental conditions, and could be easily detected on cells in pure culture or in clinical samples from humans and experimentally infected hamsters by fluorescent-antibody techniques. Fructose, glucose, galactose, and ribose were the major constituents of RPS. Although purified RPS was not immunogenic in rabbits, it was presumed to be a major antigen of the cell because it could specifically absorb approximately one-third of the antibody nitrogen in antisera prepared against whole cells of R. dentocariosa. Haptene inhibition studies indicated that fructose was the principal determinant of serological specificity in RPS. This polysaccharide was found to be serologically unique and did not cross-react with the polysaccharides and surface polymers of other oral actinomycetes and filamentous organisms.