Streptococci that produced acetoin and alkaline phosphatase, hydrolyzed arginine, and fermented trehalose but did not produce pyrrolidonylarylamidase or P-glucuronidase, split hippurate, or ferment ribose or glycogen were collected and compared, These streptococci were considered members of the unapproved species 'LStreptococcus millen'," but most of them would fit the description of one of three approved species: Streptococcus anginosus, Streptococcus constellatus, or Streptococcus intermedius. Most were recent clinical isolates. Some hydrolyzed esculin and fermented lactose, while others did pot. Some fermented mannitol and rafknose. Many were beta-hemolytic, and several reacted to antiserum of Lancefield group A, C, F, or G. From a total of 111 strains, 40 were selected for comparison of their deoxyribbnucleic acid (DNA) base sequence similarities by DNA-DNA hybridization on membrane Filters. All biotypes, hemolytic types, and serotypes were included, as well as the type strains of S. anginosus, S. constellatus, and S . intermedius, and Lancefield group F Streptococcus sp., plus two strains derived from GuthoPs '(S. millen'" isolates. The results showed considerable genetic similarity within the group. DNA from most strains hybridized at a level of 70% or more of the homologous control, even under very stringent conditions. There was somewhat less homology between DNAs of some of the least reactive strains (lactose, mannitol, and esculin negative) and the most reactive strains (lactose, mannitol, and esculin positive). The Lancefield F strain and the type strains of S. anginosus and S.constellatus were genetically similar. Traits such as hemolysis and lactose fermentation could not be ascribed to plasmids. The results support the unification of these streptococci into a single species.

The genetic relationships among 33 streptococci that were identified as Streptococcus salivarius or Streptococcus bovis and were isolated from humans were determined by deoxyribonucleic aciddeoxyribonucleic acid hybridization on membrane filters. The phenotypic characteristics of these bacteria were determined by their action on 20 substrates in a commercially prepared system, as well as by colony morphology and conventional tests. The S . sulivurius strains were biochemically heterogeneous but genetically homogeneous. Although there were some phenotypic similarities between S . bovis and S . salivarius, these two species were genetically distinct. Within S. bovis there was genetic heterogeneity. Typical S . bovis strains (S. bovis biotype I) were genetically homologous with some, but not all, of the S . bovis variant strains ( S . bovis biotype 11). Other S . bovis biotype I1 strains formed a separate genetic group, the members of which were biochemically somewhat different from other S . bovis biotype I1 strains.Streptococcus bovis has become medically important since it was discovered to be a frequent cause of endocarditis (13,19,20,30,35) and to be more common in the bowels of patients with colon diseases, including carcinomas (23 , 24,31,36). Isolates from such human sources ferment mannitol, usually ferment inulin, and produce an extracellular glucan from sucrose (13, 34). These typical strains are called Streptococcus bovis biotype I to differentiate them from strains of a variant type called Streptococcus bovis biotype 11, which do not ferment mannitol or inulin and do not produce glucan (13, 34). As is usual with such divisions, there are strains whose characteristics put them midway between the biotypes (for example, mannitol fermentation but no glucan production). In addition, some members of the common human commensal Streptococcus salivarius resemble Streptococcus bovis because they have similar biochemical traits and may react with Lancefield group D antiserum or grow on bile-esculin agar (37) or both. Like Streptococcus bovis, Streptococcus salivarius produces a polymer, usually levan (21, 32), from sucrose, and the two species can produce similar colonies on sucrose agar.To clarify the relationships among strains identified as Streptococcus salivarius, Streptococcus bovis biotype I, andStreptococcus bovis biotype 11, we compared the base sequence similarities of their deoxyribonucleic acids (DNAs) by using DNA-DNA hybridization. MATERIALS AND METHODSThe strains used in this study are listed in the tables (see below). Strains whose designations begin with CDC were obtained from the Centers for Disease Control, Atlanta, Ga. ; strains whose designations begin with COL were obtained from the Central Public Health Laboratories, Colindale, England; and strains whose designations begin with MG were obtained from The Massachusetts General Hospital, Boston. Other strains were obtained from clinics at the University of Connecticut Health Center, Farmington. Strain 13419 was a subculture of Streptococcus sa...

The guanine-plus-cytosine contents of Rothia dentocariosa strains ATCC 17931T (T = type strain) and ATCC 19426 were determined by thermal denaturation and found to be 54 and 57 mol%, respectively. These values are lower than the values given in many publications, but are consistent with data cited in some other references.The guanine-plus-cytosine (G + C) content of Rothia dentocariosa was reported by Hammond (4) to be 65.4 to 69.7 mol%, and these values are included in the description of the species in Bergey's Manual of Determinative Bacteriology, 8th ed. (1). These base contents are given in later publications which cite Hammond directly (8) or indirectly (2, 3). However, Slack and Gerencser (10) published lower figures, 47 to 53 mol% G+C, for five strains and cited unpublished data from Hammond. These data were cited by Schaal and Pulverer (9).To resolve this discrepancy, we determined the G+C contents of two American Type Culture Collection strains of R. dentocariosa and one clinical isolate. Strains ATCC 17931T (T = type strain) and ATCC 19426 were obtained from the American Type Culture Collection and were grown on casein soy agar supplemented with 10% sheep blood and in brai'n heart infusion broth. Small broth cultures were used to inoculate 100-ml portions of brain heart infusion broth, which were then used to start l-liter cultures. All cultures were tested for purity by streaking onto blood agar plates and by Gram staining. The characteristic large white colonies and pleomorphic cells were observed.The growth of l-liter cultures was monitored turbidimetrically for about 26 h. Then 10,000 U of penicillin G (10 pg/ml) was added, and the cultures were incubated for an additional 1 h and harvested. The cells were suspended in 15 ml of 0.015 M NaCl and incubated with 10 mg of lysozyme (Sigma Chemical Co., St. Louis, Mo.) for 1 h at 37°C and then with 200 U of mutanolysin (Sigma) at 55°C for 1 h. The suspension was made 0.15 M NaC1-0.0015 M sodium citrate and heated to 60°C, and the cells were lysed by adding 1 ml of 25% (wt/vol) sodium dodecyl sulfate. The deoxyribonucleic acid (DNA) was purified by phenol and chloroform deproteinizations, treatment with ribonuclease, and precipitation with ethanol (6). The yields of DNA from these slowly growing, lysis-resistant bacteria were small. About 0.5 mg was isolated from each l-liter culture. The values for absorbance of ultraviolet light by this DNA at 260, 230, and 280 nm had a ratio of 1:0.56:0.57, indicating that there was some residual carbohydrate but very little protein (6).The G+C contents were determined by thermal denaturation in 0.15 M NaCI-0.015 M sodium citrate, using the method and formula of Marmur and Doty (7). The temperature was raised OS"C/min by an automatic cuvette heater (Gilford Instrument Laboratories, Inc., Oberlin, Ohio) while the absorbance was continually recorded. The system was calibrated by simultaneous denaturation of DNA from Escherichia coli strain K-12 (51.5 mol% G+C [5]). The tempera-* Corresponding author. ture of the cuvette ...