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

Abstract

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...

Evaluation of Several Biochemical and Molecular Techniques for Identification of Streptococcus pneumoniae and Streptococcus pseudopneumoniae and Their Detection in Respiratory Samples

“…Several previous studies showed that a PCR assay targeting the Spn9802 fragment detects only S. pneumoniae and S. pseudopneumoniae but not S. mitis (4,10,32). False-positive results with Spn9802 primers have been described, and therefore, it remains unknown whether the Spn9802 DNA fragment is specific for S. pneumoniae and S. pseudopneumoniae or whether specificity is obtained by particular signatures (26).…”

“…The real-time PCR assays targeting the Spn9802 and lytA fragments were adapted from methods described previously by Suzuki et al (32) and Carvalho et al (7). The primers and probes for the inhibition and extraction control phocine herpesvirus (PhHV) were described previously (34).…”

“…S. pneumoniae belongs to the mitis group of streptococci, and several molecular assays have been applied in an attempt to discriminate S. pneumoniae from other mitis group streptococci. A variety of genes has been used as target for a (real-time) PCR assay to detect S. pneumoniae, including genes encoding the virulence factors autolysin (lytA) and pneumolysin (ply) and the DNA fragment Spn9802 (1,4,7,16,22,25,32). However, most of these PCR assays are not able to discriminate S. pneumoniae from the recently discovered Streptococcus pseudopneumoniae and other mitis group streptococci.…”

“…The biochemical assays that were used were bile solubility testing by classical tube bile solubility and oxgall disk and optochin susceptibility testing upon incu-bation in CO 2 and O 2 atmospheres. The molecular assays used were MALDI-TOF MS, real-time PCR assays targeting the Spn9802 fragment and the lytA gene, and sequence analysis of the tuf and rpoB genes (7,9,28,32,35). In addition, the presence of S. pneumoniae and S. pseudopneumoniae was determined in the respiratory specimens from which the isolates of the collection were isolated.…”

Development of Strain-Specific PCR Primers for Quantitative Detection of <i>Bacillus mesentericus</i> Strain TO-A in Human Feces

“…A number of useful polymorphic techniques, such as RAPD, repetitive-sequence-based PCR (rep-PCR), enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), and amplified fragment length polymorphism (AFLP), have been used for the detection of organism-specific distinctive DNA sequences. [28][29][30][31][32][33][34] Although only a few reports describe specific genetic detection methods applicable at the strain level, Fujimoto et al, 35) Maruo et al, 36) and Tilsala-Timisjärvi & Alatossava 37) reported the use of RAPD for strain-specific detection of Lactococcus lactis ssp. cremoris FC, Lactobacillus casei Shirota, and Lactobacillus rhamnosus Lc 1/3, respectively.…”