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Overview. We required that several criteria be met by the strain of S. sanguinis selected for genomic sequencing. First, its biotype must be in keeping with the features that define S. sanguinis using accepted diagnostic tests. Second the strain must be able to aggregate human blood platelets. Third, it must be genetically transformable. Fourth, it must be able to promote co-aggregation with other bacteria in patterns consistent with S. sanguinis. Fifth, it must be virulent in the rat model of infective endocarditis.

After preliminary characterization and consideration of several laboratory strains and fresh clinical isolates, we focused our attention on three strains previously identified and studied by Mogens Kilian and co-workers. These strains, designated SK36, SK85 and SK158, were studied by our group and by collaborators. Based on these studies have selected strain SK36 for genomic sequencing. S. sanguinis SK36 was isolated from human dental plaque by Mogens Kilian in 1980 as part of a study described in Kilian and Holmgren, (Infection and Immunity 31:868-873, 1981), and is listed in a taxonomic study reported by the Kilian group (Kilian et al. Int. J. Sys.t Bacteriol .39:471-484, 1989) as biovar 1 of S. sanguinis. The strain was also used in the study that the Kilian group did on the adhesive properties of streptococci with John Cisar and his co-workers (Hsu et al. Microbial Ecology in Health and Disease 7: 125-137, 1994).

Diagnostic criteria. S. sanguinis SK36 was kindly evaluated by Dr. Lisa Weymouth, director of the clinical microbiology laboratory at MCV Hospitals at Virginia Commonwealth University. Using the api20Strep™ System (bioMerieux), strain SK36 was confirmed as Streptococcus sanguinis according to key biotype characteristics. SK36 hydrolyzed esculin, produced alpha-galactosidase and leucine arylamidase, and it produced acid from lactose, trehalose, and raffinose. It weakly acidified starch.

We also assayed culture supernatants of SK36 for the presence of glucosyltransferase (GTF) activity . This key characteristic of S. sanguinis is not evaluated by any diagnostic test in the clinical setting. Using the standard assay for glucosyltransferase (Dertzbaugh M.T. and F. L. Macrina . Infect Immun 58:1509-13, 1990) we evaluated GTF activity of strain SK36 using Streptococcus mutans GS-5 as a control. This latter strain produces multiple GTF enzymes, that synthesize both water-soluble and water-insoluble glucan polymers. The results of these assays were as follows:

Cells were grown to OD₆₆₀ of 0.07 in Todd Hewitt broth containing 2.5% heat-inactivated horse serum. DNA was added, the incubation continued for one hour at 37°C, and the cells plated on trypticase soy agar containing 10 µg/ml erythromycin.

^† CSP is (200 ng/ml) Competence Stimulating Peptide from S. sanguinis (strain 13b). Provided by Peter Gaustad, Oslo, Norway. (Gaustad, P., and L. S. Havardstein. 1997. Competence-pheromone in Streptococcus sanguinis. Identification of the competence gene comC and the competence pheromone. Adv Exp Med Biol. 418:1019-21.)

Co-aggregation studies. Dr. Paul Kolenbrander and Ms. Roxanna Andersen kindly evaluated the ability of S. sanguinis SK36 to co-aggregate with a variety of oral bacteria. Its cooaggregation scores were indistinguishable from those obtained using S. sanguinis 10556, the type strain of this species. SK 36 coaggregated with: Streptococcus oralis 34, Streptococcus oralis C104 and Streptococcus SM PK509, Actinomyces naeslundii PK19, Actinomyces naeslundii PK947, and Actinomyces naeslundii PK606. It also coaggregated with Fusobacterium nucleatum subsp. nucleatum PK1594, Fusobacterium nucleatum subsp. vincentii ATCC49256, and Fusobacterium nucleatum subsp. polymorphum ATCC10953.

Virulence. We tested SK36 and SK158 in the rat model of endocarditis using standard methods (Munro, C. and F. Macrina. Sucrose-derived exopolysaccharides of Streptococcus mutans V403 contribute to infectivity in endocarditis.. Mol Microbiol 1993 8:133-42). S. sanguinis SK36 showed infectivity levels that were comparable to other virulent strains.

Conclusion. Based on the foregoing observations we selected S. sanguinis SK36 for genomic sequencing.