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Journal of Clinical Microbiology, December 2005, p. 6073-6085, Vol. 43, No. 12
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.12.6073-6085.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Use of Phylogenetic and Phenotypic Analyses To Identify Nonhemolytic Streptococci Isolated from Bacteremic Patients

Tomonori Hoshino,1,2 Taku Fujiwara,2 and Mogens Kilian1*

Institute of Medical Microbiology and Immunology, Aarhus University, Bartholin Building, DK-8000 Aarhus C, Denmark,1 Division of Pediatric Dentistry, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan2

Received 22 July 2005/ Returned for modification 6 September 2005/ Accepted 4 October 2005

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.

* Corresponding author. Mailing address: Institute of Medical Microbiology and Immunology, Aarhus University, Bartholin Building, DK-8000 Aarhus C, Denmark. Phone: 45-8942-1735. Fax: 45-8619-6128. E-mail: kilian{at}microbiology.au.dk.

Journal of Clinical Microbiology, December 2005, p. 6073-6085, Vol. 43, No. 12
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.12.6073-6085.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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