Two oligonucleotide primer sets for the discrimination of Streptococcus pneumoniae from "pneumococcuslike" 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.Streptococcus pneumoniae is a major cause of bacterial disease in humans, including pneumonia, otitis media, septicemia, and meningitis. Two naturally transformable viridans group streptococci, Streptococcus mitis and Streptococcus oralis, are closely related to S. pneumoniae, and classification of these organisms has long been considered difficult (32). Indeed, the nucleotide sequences of the 16S rRNA genes from S. mitis and S. oralis are over 99% identical to that of S. pneumoniae (17). As the potentially pathogenic S. pneumoniae is frequently detected in the oral cavity (31), and the commensals S. mitis and S. oralis are almost always detected (18), it is important to accurately discriminate among them in order to provide accurate diagnoses and treatments.Various molecular assays have been employed to identify pneumococcal strains and to detect pneumococci directly from clinical samples. PCR-based assays for identifying S. pneumoniae have frequently targeted genes encoding pneumococcal virulence factors, including pneumolysin (ply) (7), autolysin (lytA) (20), pneumococcal surface antigen A (21), manganesedependent superoxide dismutase (sodA) (16), and penicillin binding protein (24). Ideally, amplification of these target genes should be specific for S. pneumoniae isolates only. However, the occasional occurrence of viridans group identified as S. mitis or S. oralis harbor genes encoding S. pneumoniae virulence factors has been reported (31).In this study, genomic subtractive hybridization was performed to identify genomic differences between the two type strains, S. pneumoniae WU2...

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It was previously reported that two oligonucleotide primer sets (spn9802 and spn9828) for discriminating Streptococcus pneumoniae from pneumococcus-like oral streptococcal isolates using PCR had been developed. In this study, PCR amplification of the lytA, ply, spn9802 and spn9828 genes was used to identify presumptive S. pneumoniae. Two genetic groups were identified by analysing sputum samples from 28 patients with community-acquired pneumonia: the lytA-positive, ply-positive, spn9802-positive and spn9828-negative group, and the lytA-positive, ply-positive, spn9802-positive and spn9828-positive group. Isolates of the former group were resistant to optochin, while those of the latter group showed susceptibility to optochin. The lytA-positive, ply-positive, spn9802-negative and spn9828-negative isolates, and lytA-positive, ply-positive, spn9802-negative and spn9828-positive isolates, were not detected in sputum from patients with pneumonia. Subsequently, a total of 92 saliva samples from healthy individuals was screened by PCR using these primer sets. The lytA-positive, ply-positive, spn9802-positive and spn9828-negative group was identified more frequently in saliva from healthy children than in saliva from older healthy individuals and patients with pneumonia. The lytA-positive, ply-positive, spn9802-positive and spn9828-positive group was found frequently in saliva from healthy children, and in saliva and sputum from patients with pneumonia. This study demonstrates a rapid, optimal screening method for the genotypic identification of presumptive S. pneumoniae by PCR using four genes highly specific for S. pneumoniae.

The cytokine IL-33 is constitutively expressed in epithelial cells and it augments Th2 cytokine-mediated inflammatory responses by regulating innate immune cells. We aimed to determine the role of the periodontal pathogen, Porphyromonas gingivalis, in the enhanced expression of IL-33 in human gingival epithelial cells. We detected IL-33 in inflamed gingival epithelium from patients with chronic periodontitis, and found that P. gingivalis increased IL-33 expression in the cytoplasm of human gingival epithelial cells in vitro. In contrast, lipopolysaccharide, lipopeptide, and fimbriae derived from P. gingivalis did not increase IL-33 expression. Specific inhibitors of P. gingivalis proteases (gingipains) suppressed IL-33 mRNA induction by P. gingivalis and the P. gingivalis gingipain-null mutant KDP136 did not induce IL-33 expression. A small interfering RNA for protease-activated receptor-2 (PAR-2) as well as inhibitors of phospholipase C, p38 and NF-κB inhibited the expression of IL-33 induced by P. gingivalis. These results indicate that the PAR-2/IL-33 axis is promoted by P. gingivalis infection in human gingival epithelial cells through a gingipain-dependent mechanism.

A TaqMan PCR was developed for quantifying early colonizer microorganisms in dental biofilms. To design species-specific primers and TaqMan probes, genomic subtractive hybridization was used. This quantitative assay in combination with subtractive hybridization may be of value in the study of microbial ecosystems consisting of related species that are involved in the formation and etiology of biofilms.