The aim of this study was to evaluate molecular and phenotypic methods for the identification of nonhemolytic streptococci. A collection of 148 strains consisting of 115 clinical isolates from cases of infective endocarditis, septicemia, and meningitis and 33 reference strains, including type strains of all relevant Streptococcus species, were examined. Identification was performed by phylogenetic analysis of nucleotide sequences of four housekeeping genes, ddl, gdh, rpoB, and sodA; by PCR analysis of the glucosyltransferase (gtf) gene; and by conventional phenotypic characterization and identification using two commercial kits, Rapid ID 32 STREP and STREPTOGRAM and the associated databases. A phylogenetic tree based on concatenated sequences of the four housekeeping genes allowed unequivocal differentiation of recognized species and was used as the reference. Analysis of single gene sequences revealed deviation clustering in eight strains (5.4%) due to homologous recombination with other species. This was particularly evident in S. sanguinis and in members of the anginosus group of streptococci. The rate of correct identification of the strains by both commercial identification kits was below 50% but varied significantly between species. The most significant problems were observed with S. mitis and S. oralis and 11 Streptococcus species described since 1991. Our data indicate that identification based on multilocus sequence analysis is optimal. As a more practical alternative we recommend identification based on sodA sequences with reference to a comprehensive set of sequences that is available for downloading from our server. An analysis of the species distribution of 107 nonhemolytic streptococci from bacteremic patients showed a predominance of S. oralis and S. anginosus with various underlying infections.The genus Streptococcus currently consists of more than 50 species, most of which belong to one of six phylogenetic clusters that are revealed by comparative analysis of 16S rRNA gene sequences. In addition to the pyogenic group, which includes the traditional pathogenic species (i.e., hemolytic streptococci), these clusters are the anginosus group, the mitis group, the salivarius group, the bovis group, and the mutans group (30,34). Many of the species of these five clusters are major constituents of the commensal microbiota of the human oral cavity and upper respiratory tract and are occasionally implicated in various pathologies. The anginosus group, formerly called "Streptococcus milleri" in some parts of the world (16), includes three recognized species (Streptococcus anginosus, Streptococcus intermedius, and Streptococcus constellatus) that are primarily associated with suppurative infections of tissues of the mouth and various body sites, including the meninges (9,37,44,54,56). The mitis group currently includes

Streptococcus mutans produces 3 types of glucosyltransferase (GTF), whose cooperative action is considered to be essential for its cellular adherence to the tooth surface. However, the precise mechanisms for synthesizing adhesive glucans and the specific roles of each GTF in cellular adherence to smooth surfaces have not been elucidated. In the present study, seven types of isogenic mutants of S. mutans MT8148 lacking GTFB, GTFC, and/or GTFD activities were constructed by inactivation of the genes encoding GTFB, GTFC, and/or GTFD. Furthermore, recombinant GTFB, GTFC, and GTFD were prepared from Escherichia coli cells harboring recombinant plasmids containing each of the gtf genes. Using these GTF-deficient mutants and rGTFs, we reconstituted sucrose-dependent adherence of S. mutans resting cells and examined the role of each GTF in vitro. The highest level of sucrose-dependent adherence was found at the ratio of 20 rGTFB:1 rGTFC:4 rGTFD in both the resting cells of GTF-deficient mutants and insoluble glucan synthesized by rGTFs. Moreover, when rGTFC and rGTFD were both present at concentrations of 1.5 mU and 6 mU, respectively, the insoluble glucan synthesized from sucrose by the rGTFs showed a high level of adhesiveness to smooth surfaces, even without rGTFB. These results suggest that the presence of all three GTFs at the optimum ratio is necessary for sucrose-dependent adherence of S. mutans, and that GTFC and GTFD may play significant roles in the synthesis of adhesive and insoluble glucan from sucrose.

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We present systematic total energy calculations for metals ͑Al, Fe, Ni, Cu, Rh, Pd, and Ag͒ and semiconductors ͑C, Si, Ge, GaAs, InSb, ZnSe, and CdTe͒, based on the all-electron full-potential ͑FP͒ Korringa-KohnRostoker Green's-function method, using density-functional theory. We show that the calculated lattice parameters and bulk moduli are in excellent agreement with calculated results obtained by other FP methods, in particular, the full-potential linear augmented-plane-wave method. We also investigate the difference between the local-spin-density approximation ͑LSDA͒ and the generalized-gradient approximation ͑GGA͒ of Perdew and Wang ͑PW91͒, and find that the GGA corrects the deficiencies of the LSDA for metals, i.e., the underestimation of equilibrium lattice parameters and the overestimation of bulk moduli. On the other hand, for semiconductors the GGA gives no significant improvement over the LSDA. We also discuss that a perturbative GGA treatment based on FP-LSDA spin densities gives very accurate total energies. Further, we demonstrate that the accuracy of structural properties obtained by FP-LSDA and FP-GGA calculations can also be achieved in the calculations with spherical potentials, provided that the full spin densities are calculated and all Coulomb and exchange integrals over the Wigner-Seitz cell, occurring in the double-counting contributions of the total energy, are correctly evaluated. ͓S0163-1829͑99͒15331-1͔