1887

Abstract

The need for a microbial identification of independent of culture methods has resulted in the introduction of other laboratory principles. The verification of a proper and exclusive gene for the detection of the pneumococcus by nucleic acid-based tests is, however, still unresolved. A previously published -gene-specific real-time PCR method was applied to a panel of bacterial strains to clarify the analytical sensitivity and specificity of a PCR assay targeting this gene. Furthermore, a phylogenetic analysis of published gene sequences was performed to look at gene sequence differences and the theoretical match with the primers and probes. The -gene-specific PCR detected 46/46 isolates. All 49 of the non-pneumococcal isolates tested negative, including 22 isolates from the group streptococci. Phylogenetic analysis of 94 sequences of the gene from different strains of , and showed that 70/87 sequences constituted one cluster and a further six sequences were outside but adjacent to this cluster, all with a complete match with primers and probes. The remaining 11 strains could be placed in a different cluster, which also contained the five and two strains. All strains had no match with primers and probes. The strains in the second cluster were all characterized by being bile-insoluble, an infrequent pneumococcal phenotype. Routine laboratories can utilize the additional observation that pneumococci that were negative by the specific PCR also carried the phenotype of bile insolubility, thereby observing the incidence of false-negative results produced by the PCR assay. The real-time PCR targeting the gene thus constitutes a sensitive and specific assay that distinguishes from its close relatives in the group.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.036574-0
2012-04-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jmm/61/4/478.html?itemId=/content/journal/jmm/10.1099/jmm.0.036574-0&mimeType=html&fmt=ahah

References

  1. Arbique J. C., Poyart C., Trieu-Cuot P., Quesne G., Carvalho M. G., Steigerwalt A. G., Morey R. E., Jackson D., Davidson R. J., Facklam R. R. 2004; Accuracy of phenotypic and genotypic testing for identification of Streptococcus pneumoniae and description of Streptococcus pseudopneumoniae sp. nov. J Clin Microbiol 42:4686–4696 [View Article][PubMed]
    [Google Scholar]
  2. Carvalho M. G., Steigerwalt A. G., Thompson T., Jackson D., Facklam R. R. 2003; Confirmation of nontypeable Streptococcus pneumoniae-like organisms isolated from outbreaks of epidemic conjunctivitis as Streptococcus pneumoniae. J Clin Microbiol 41:4415–4417 [View Article][PubMed]
    [Google Scholar]
  3. Carvalho M. G., Tondella M. L., McCaustland K., Weidlich L., McGee L., Mayer L. W., Steigerwalt A., Whaley M., Facklam R. R. other authors 2007; Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA. J Clin Microbiol 45:2460–2466 [View Article][PubMed]
    [Google Scholar]
  4. Corless C. E., Guiver M., Borrow R., Edwards-Jones V., Fox A. J., Kaczmarski E. B. 2001; Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. J Clin Microbiol 39:1553–1558 [View Article][PubMed]
    [Google Scholar]
  5. Donati C., Hiller N. L., Tettelin H., Muzzi A., Croucher N. J., Angiuoli S. V., Oggioni M., Dunning Hotopp J. C., Hu F. Z. other authors 2010; Structure and dynamics of the pan-genome of Streptococcus pneumoniae and closely related species. Genome Biol 11:R107 [View Article][PubMed]
    [Google Scholar]
  6. Garcia J. L., Diaz E., Romero A., Garcia P. 1994; Carboxy-terminal deletion analysis of the major pneumococcal autolysin. J Bacteriol 176:4066–4072[PubMed]
    [Google Scholar]
  7. Hanage W. P., Kaijalainen T., Herva E., Saukkoriipi A., Syrjänen R., Spratt B. G. 2005; Using multilocus sequence data to define the pneumococcus. J Bacteriol 187:6223–6230 [View Article][PubMed]
    [Google Scholar]
  8. Kee C., Palladino S., Kay I., Pryce T. M., Murray R., Rello J., Gallego M., Lujan M., Muñoz-Almagro C., Waterer G. W. 2008; Feasibility of real-time polymerase chain reaction in whole blood to identify Streptococcus pneumoniae in patients with community-acquired pneumonia. Diagn Microbiol Infect Dis 61:72–75 [View Article][PubMed]
    [Google Scholar]
  9. Kilian M., Poulsen K., Blomqvist T., Håvarstein L. S., Bek-Thomsen M., Tettelin H., Sørensen U. B. 2008; Evolution of Streptococcus pneumoniae and its close commensal relatives. PLoS ONE 3:e2683 [View Article][PubMed]
    [Google Scholar]
  10. Llull D., López R., García E. 2006; Characteristic signatures of the lytA gene provide a basis for rapid and reliable diagnosis of Streptococcus pneumoniae infections. J Clin Microbiol 44:1250–1256 [View Article][PubMed]
    [Google Scholar]
  11. Mosser J. L., Tomasz A. 1970; Choline-containing teichoic acid as a structural component of pneumococcal cell wall and its role in sensitivity to lysis by an autolytic enzyme. J Biol Chem 245:287–298[PubMed]
    [Google Scholar]
  12. Obregón V., García P., García E., Fenoll A., López R., García J. L. 2002; Molecular peculiarities of the lytA gene isolated from clinical pneumococcal strains that are bile insoluble. J Clin Microbiol 40:2545–2554 [View Article][PubMed]
    [Google Scholar]
  13. Richter S. S., Heilmann K. P., Dohrn C. L., Riahi F., Beekmann S. E., Doern G. V. 2008; Accuracy of phenotypic methods for identification of Streptococcus pneumoniae isolates included in surveillance programs. J Clin Microbiol 46:2184–2188 [View Article][PubMed]
    [Google Scholar]
  14. Sheppard C. L., Harrison T. G., Morris R., Hogan A., George R. C. 2004; Autolysin-targeted LightCycler assay including internal process control for detection of Streptococcus pneumoniae DNA in clinical samples. J Med Microbiol 53:189–195 [View Article][PubMed]
    [Google Scholar]
  15. Strålin K., Bäckman A., Holmberg H., Fredlund H., Olcén P. 2005; Design of a multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae to be used on sputum samples. APMIS 113:99–111 [View Article][PubMed]
    [Google Scholar]
  16. Suzuki N., Seki M., Nakano Y., Kiyoura Y., Maeno M., Yamashita Y. 2005; Discrimination of Streptococcus pneumoniae from viridans group streptococci by genomic subtractive hybridization. J Clin Microbiol 43:4528–4534 [View Article][PubMed]
    [Google Scholar]
  17. Suzuki N., Yuyama M., Maeda S., Ogawa H., Mashiko K., Kiyoura Y. 2006; Genotypic identification of presumptive Streptococcus pneumoniae by PCR using four genes highly specific for S. pneumoniae. J Med Microbiol 55:709–714 [View Article][PubMed]
    [Google Scholar]
  18. Tamura K., Dudley J., Nei M., Kumar S. 2007; mega4: Molecular Evolutionary Genetics Analysis (mega) software version 4.0. Mol Biol Evol 24:1596–1599 [View Article][PubMed]
    [Google Scholar]
  19. Whatmore A. M., Efstratiou A., Pickerill A. P., Broughton K., Woodard G., Sturgeon D., George R., Dowson C. G. 2000; Genetic relationships between clinical isolates of Streptococcus pneumoniae, Streptococcus oralis, and Streptococcus mitis: characterization of “atypical” pneumococci and organisms allied to S. mitis harboring S. pneumoniae virulence factor-encoding genes. Infect Immun 68:1374–1382 [View Article][PubMed]
    [Google Scholar]
  20. Zbinden A., Köhler N., Bloemberg G. V. 2011; recA-based PCR assay for accurate differentiation of Streptococcus pneumoniae from other viridans streptococci. J Clin Microbiol 49:523–527 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.036574-0
Loading
/content/journal/jmm/10.1099/jmm.0.036574-0
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF

Supplementary File 1

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error