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Journal of Clinical Microbiology, September 2005, p. 4528-4534, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4528-4534.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Discrimination of Streptococcus pneumoniae from Viridans Group Streptococci by Genomic Subtractive Hybridization

Nao Suzuki,1 Mitsuko Seki,2,3 Yoshio Nakano,4 Yusuke Kiyoura,1 Masao Maeno,2,3 and Yoshihisa Yamashita4*

Department of Oral Medical Science, Ohu University School of Dentistry, 31-1 Misumido, Tomitamachi, Koriyama 963-8611, Japan,1 Department of Oral Health Sciences, Nihon University, School of Dentistry, 1-8-13 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-8310, Japan,2 Division of Functional Morphology, Dental Research Center, Nihon University, School of Dentisry, 1-8-13 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-8310, Japan,3 Department of Preventive Dentistry, Kyushu University Faculty of Dental Science, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan4

Received 30 March 2005/ Returned for modification 16 May 2005/ Accepted 3 June 2005

Two oligonucleotide primer sets for the discrimination of Streptococcus pneumoniae from "pneumococcus-like" oral streptococcal isolates by PCR were developed. Genomic subtractive hybridization was performed to search for differences between Streptococcus pneumoniae strain WU2 and the most closely related oral streptococcus, Streptococcus mitis strain 903. We identified 19 clones that contained S. pneumoniae-specific nucleotide fragments that were absent from the chromosomal DNA of typical laboratory strains of S. mitis and other oral bacteria. Subsequently, oligonucleotide PCR primers for the detection of S. pneumoniae were designed from the sequences of the subtracted DNA fragments, and the specificities of the 19 primer sets were evaluated by PCR using chromosomal DNAs extracted from four S. pneumoniae clinical isolates and from 20 atypical organisms classified as S. mitis or S. oralis, which harbored genes encoding the pneumococcal virulence factors autolysin (lytA) or pneumolysin (ply), as templates. Of the 19 primer sets, two (Spn9802 and Spn9828) did not amplify PCR products from any of the pneumococcus-like streptococcal strains that we examined. The genes containing the Spn9802 and Spn9828 sequences encoded proteins of unknown function that did not correspond to any previously described proteins in other bacteria. These new oligonucleotide primers may be very useful for early and correct diagnosis of S. pneumoniae infections.

* Corresponding author. Mailing address: Department of Preventive Dentistry, Kyushu University Faculty of Dental Science, Fukuoka 812-8582, Japan. Phone: 81-92-642-6450. Fax: 81-92-642-6354. E-mail: yoshi{at}dent.kyushu-u.ac.jp.

Journal of Clinical Microbiology, September 2005, p. 4528-4534, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4528-4534.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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