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And Streptococcus Oralis Sp INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1982, p. 410-415 Vol. 32, No. 4 0020-7713/82/04041O-06$02 .00/0 Copyright 8 1982, International Union of Microbiological Societies Streptococcus gallinarum sp. nov. and Streptococcus oralis sp. nov. P. D. BRIDGE AND P. H. A. SNEATH Department of Microbiology, Leicester University, Leicester, LEI 7RH, England Received 22 February 1982/Accepted 2 September 1982 A numerical taxonomic survey of numerous strains of streptococci showed the presence of two clusters of strains that are distinct from the presently named species of Streptococcus. One group, from chicken intestines, is named S. gallinarum and the other, from the human mouth, is named S. oralis. The type strains are F87/276 (= PB21 = NCTC 11428) and LVG 1 (= PB182 = NCTC 11427), respectively. As the result of a numerical taxonomic survey 2 (Difco Laboratories) with the addition of 7.5% (voY of 202 strains of streptococci and closely related vol) horse blood (Difco) or on broth medium consisting organisms (P. D. Bridge, Ph.D. thesis, Universi- of (in grams per liter): proteose peptone (Difco), 15; ty of Leicester, 1981), two phenetic groups of sodium chloride, 5; yeast extract (Difco), 5; and liver digest (Oxoid Ltd.), 2.5. Acid production from carbo- strains were observed that were distinct from hydrates was observed in API 50E galleries (API those representing known species (8) of Strepto- System Ltd.) inoculated with API media and incubat- coccus. These groups were referred to as chick- ed at 35°C for 48 h. Standard methods were used for en group D and oral group 11. the following: Gram stain; hemolysis on horse blood The chicken group D strains were isolated by agar; resistance to 60°C for 15 and 60 min; growth at 4, Barnes et al. (2) while investigating the effects of 10, and 45°C; growth at pH 9.6; growth with 3 and dietary bacitracin on fecal streptococci in young 6.5% (wt/vol) NaC1, O.o004% (wt/vol) crystal violet, chicks. These strains showed properties distinct and 0.1% sodium azide; reduction of tellurite, tetrazo- from those of the established enterococci. They lium, methylene blue in milk, and nitrite; hydrolysis of were detected only after a long incubation peri- arginine, hippurate, and esculin; decarboxylation of lysine; final pH in glucose broth; dextran formation; od (8 days) on a selective medium. The oral and gelatin liquefaction (details for these, and for other group I1 strains were studied by Carlsson (3) in a tests used in the numerical taxonomy, are given by numerical taxonomy of streptococci isolated P. D. Bridge, Ph.D. thesis). from the human oral cavity. Through the kind- Growth on thallous acetate-tetrazolium agar was ness of E. M. Barnes and J. Carlsson, strains tested as described by Barnes (1) with the exception were made available for study, and the evidence that Oxoid neutralized bacteriological peptone was is presented here for considering these as two used in place of Evans peptone; cultures were incubat- new species of Streptococcus. Certain previous- ed at 35°C for 24 h and then at room temperature for 7 ly named species of the genus do not appear on days. DNA was extracted and purified by the method of the Approved Lists of Bacterial Names (ll), and Garvie (6) with minor adaptations. Melting point deter- these names are placed in quotation marks in minations were made in standard saline citrate buffer. this paper. The denaturation curves were followed with a Beck- MATERIALS METHODS man model 35 spectrophotometerwith a Stanton Red- AND croft linear temperature variable rate programmer and Bacterial strains. The 17 strains of chicken group D an Edale thermistor thermometer with a Grant Instru- and the 13 strains of oral group I1 are as follows: ments temperature probe. Chicken group D strains PB18 through PB34 are, The numerical taxonomy of the 202 strains was respectively, the strains of Barnes et al. (2) numbered based on 157 tests with the Gower coefficient and F87/273 through F87/279, F87/296, F87/307, F87/311, unweighted pair group method with averages F87/344, F87/358, F87/361, F87/362, and F87/364 (UPGMA)clustering (14). The test for distinctness of through F87/366. Oral group I1 strains PB177 through clusters was the W statistic (12); a computer program PB190 are, respectively, those of Carlsson (3) num- was used that utilizes the intercentroid distance and bered KPE2, MVEl, KPEl, LPAl, MPAl, LVGl, standard deviations along the intercentroid axis (13). OS51, LV51, NS51, OP51, NT61, PTSl, and LV81. The method permits a significance test to determine Cultural determinants. Cultures were stored at whether overlap is significantly less than that expected -80°C on glass beads (5). Strains in the laboratory from arbitrary division of a continuous variation, were grown on a basal medium of blood agar base no. referred to as a rectangular distribution (12). VOL. 32, 1982 TWO NEW SPECIES OF STREPTOCOCCUS 411 RESULTS IS 80 90 100 The strains of chicken group D clustered as a separate group at the !90% similarity level (Fig. S. foecalis la). They were most closely linked to “Strepto- coccus avium” (10). They are allied to the S. foecium classical enterococci S. faecalis and S. faecium but are not closely related to other species I-! S. avium sometimes included among the enterococci, Chicken Group D such as S. bovis and S. equinus. The chicken 4 S. gallinarum sp. nov. group D cluster is well separated from “S. a avium.” The disjunction coefficient W between %S the two clusters was 2.75, corresponding to a 80 90 100 nominal overlap of less than 0.7%. Because of the small numbers of strains in the “S. avium” S. mitis cluster, it is not possible to prove that this S. sanguis separation is statistically less than a rectangular Oral Group II one (12), for which W is close to 1.7 (or a little s. orolis sp. nov. higher when numbers of strains are small), and the overlap is about 8% (or a little lower for IS. miller; small numbers of strains). The test results, how- b ever (Table l),leave little doubt that the groups FIG. 1. Dendrograms showing the relations of the new species of Streptococcus to similar species, based are distinct. on the Gower similarity coefficient and unweighted The strains of oral group I1 formed a cluster at pair group method with averages (UPGMA)cluster- the 84% similarity level (Fig. lb). This was ing. (a) Chicken group D; (b) Oral group 11. closest to “Streptococcus milleri” (7),but the W coefficient between the two was 6.24, corre- sponding to a nominal overlap of and significantly less than that for a rectangular distribution. room temperature on thallous acetate-tetrazoli- The other species that were close were S. um agar, on which they produce pink colonies (a mitis and S. sanguis. The W value between S. property which we confirmed, Table 3). They sanguis and oral group I1 was 2.62, which indi- were of serological group D but did not react cates overlap of less than O.%, but again statis- with group Q antiserum, unlike “Streptococcus tical significance against the rectangular distri- avium” (10). Barnes et al. (2) divided their bution was not achieved because of the small isolates into three groups, Al, A2, and A3, number of strains of S. sanguis. The test results based on arginine hydrolysis and inulin fermen- (Table 2) show, however, a sufficient number of tation, but in our study there were no distinct differences to support their distinctness. The subclusters that reflected these tests. Barnes et separation of oral group I1 from S. mitis (and al. noted that cells of the strains tended to be from other oral groups such as S. mutans and S. more pointed and pleomorphic than those of S. salivarius), was highly significant. faecalis and S. faecium. The results of the physiological tests and Twenty-three tests used in this study of the DNA base ratios for the new species and those chicken group D strains are comparable to those similar to them are given in Tables 1 and 2. The used by Barnes et al. (2). Table 4 shows a results for the growth of strains of chicken group comparison of the results. The results of most D on thallous acetate-tetrazolium agar are tests are in good agreement. Barnes et al. (2) shown in Table 3. used tube methods for the production of acid from carbohydrates incubated for 7 days at 37”C,with bromcresol purple as pH indicator, DISCUSSION which only responds to acidity below about pH The distinctness of the two new species clus- 5.8. The results here are from API 50E galleries ters (shown by Fig. 1 and the overlap statistics) incubated at 37°C for 48 h, with phenol red as is as good as that for traditional species of indicator. Although the incubation time was Streptococcus. Although uniquely defining char- shorter, phenol red detects acidity at about pH acters are few or absent, this is also true for 6.8, and it may be that the higher percentage of existing species (Tables 1, 2). Neither of them positive carbohydrate reactions in this study appears to correspond to a previously named (Table 4) was due to detection of smaller species. amounts of acidity in the API galleries. The chicken group D strains were noted by The discrepancy seen in the reduction of Barnes et al. (2) because of their slow growth at methylene blue milk may be accounted for by 412 BRIDGE AND SNEATH INT. J. SYST.BACTERIOL. TABLE 1. Test results for S.
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