Discrimination of Streptococcus pneumoniae from viridans group streptococci by genomic subtractive hybridization.
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.
Suzuki
Nao
N
Department of Oral Medical Science, School of Dentistry, Tomitamachi, Koriyama, Japan.
Seki
Mitsuko
M
Nakano
Yoshio
Y
Kiyoura
Yusuke
Y
Maeno
Masao
M
Yamashita
Yoshihisa
Y
D001426:Bacterial Proteins / Q000235:genetics; D015373:Bacterial Typing Techniques; D002648:Child; D002675:Child, Preschool; D004269:DNA, Bacterial / Q000032:analysis*; D006801:Humans; D009061:Mouth Mucosa / Q000382:microbiology; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D013045:Species Specificity; D034361:Streptococcus mitis / Q000145:classification / Q000235:genetics; D019533:Streptococcus oralis / Q000145:classification / Q000235:genetics; D013296:Streptococcus pneumoniae / Q000145:classification* / Q000235:genetics; D013301:Streptolysins / Q000235:genetics; D034363:Viridans Streptococci / Q000145:classification* / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae; D009238:N-Acetylmuramoyl-L-alanine Amidase