1887

Journal of Medical Microbiology logo

Volume 61, Issue 11

Identification of bacteria directly from positive blood culture samples by DNA pyrosequencing of the 16S rRNA gene Free

Abstract

Rapid identification of the causative bacteria of sepsis in patients can contribute to the selection of appropriate antibiotics and improvement of patients’ prognosis. Genotypic identification is an emerging technology that may provide an alternative method to, or complement, established phenotypic identification procedures. We evaluated a rapid protocol for bacterial identification based on PCR and pyrosequencing of the V1 and V3 regions of the 16S rRNA gene using DNA extracted directly from positive blood culture samples. One hundred and two positive blood culture bottles from 68 patients were randomly selected and the bacteria were identified by phenotyping and pyrosequencing. The results of pyrosequencing identification displayed 84.3 and 64.7 % concordance with the results of phenotypic identification at the genus and species levels, respectively. In the monomicrobial samples, the concordance between the results of pyrosequencing and phenotypic identification at the genus level was 87.0 %. Pyrosequencing identified one isolate in 60 % of polymicrobial samples, which were confirmed by culture analysis. Of the samples identified by pyrosequencing, 55.7 % showed consistent results in V1 and V3 targeted sequencing; other samples were identified based on the results of V1 (12.5 %) or V3 (31.8 %) sequencing alone. One isolate was erroneously identified by pyrosequencing due to high sequence similarity with another isolate. Pyrosequencing identified one isolate that was not detected by phenotypic identification. The process of pyrosequencing identification can be completed within ~4 h. The information provided by DNA-pyrosequencing for the identification of micro-organisms in positive blood culture bottles is accurate and could prove to be a rapid and useful tool in standard laboratory practice.

  • Received:
  • Accepted:
  • Published Online:
© 2012 SGM
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.049163-0
2012-11-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/jmm/61/11/1556.html?itemId=/content/journal/jmm/10.1099/jmm.0.049163-0&mimeType=html&fmt=ahah

References

  1. Barenfanger J., Drake C., Kacich G. 1999; Clinical and financial benefits of rapid bacterial identification and antimicrobial susceptibility testing. J Clin Microbiol 37:1415–1418[PubMed]
     [Google Scholar]
  2. Barenfanger J., Short M. A., Groesch A. A. 2001; Improved antimicrobial interventions have benefits. J Clin Microbiol 39:2823–2828 [View Article][PubMed]
     [Google Scholar]
  3. Bosshard P. P., Zbinden R., Abels S., Böddinghaus B., Altwegg M., Böttger E. C. 2006; 16S rRNA gene sequencing versus the API 20 NE system and the VITEK 2 ID-GNB card for identification of nonfermenting Gram-negative bacteria in the clinical laboratory. J Clin Microbiol 44:1359–1366 [View Article][PubMed]
     [Google Scholar]
  4. Christner M., Rohde H., Wolters M., Sobottka I., Wegscheider K., Aepfelbacher M. 2010; Rapid identification of bacteria from positive blood culture bottles by use of matrix-assisted laser desorption-ionization time of flight mass spectrometry fingerprinting. J Clin Microbiol 48:1584–1591 [View Article][PubMed]
     [Google Scholar]
  5. Downes A., Thornhill C., Mallard R. H. 1998; An evaluation of Vitek – an automated system for bacterial identification and susceptibility testing. Commun Dis Public Health 1:206–207[PubMed]
     [Google Scholar]
  6. Jonasson J., Olofsson M., Monstein H. J. 2002; Classification, identification and subtyping of bacteria based on pyrosequencing and signature matching of 16S rDNA fragments. APMIS 110:263–272 [View Article][PubMed]
     [Google Scholar]
  7. Jordan J. A., Butchko A. R., Durso M. B. 2005; Use of pyrosequencing of 16S rRNA fragments to differentiate between bacteria responsible for neonatal sepsis. J Mol Diagn 7:105–110 [View Article][PubMed]
     [Google Scholar]
  8. Jordan J. A., Jones-Laughner J., Durso M. B. 2009; Utility of pyrosequencing in identifying bacteria directly from positive blood culture bottles. J Clin Microbiol 47:368–372 [View Article][PubMed]
     [Google Scholar]
  9. Kolbert C. P., Persing D. H. 1999; Ribosomal DNA sequencing as a tool for identification of bacterial pathogens. Curr Opin Microbiol 2:299–305 [View Article][PubMed]
     [Google Scholar]
  10. Luna R. A., Fasciano L. R., Jones S. C., Boyanton B. L. Jr, Ton T. T., Versalovic J. 2007; DNA pyrosequencing-based bacterial pathogen identification in a pediatric hospital setting. J Clin Microbiol 45:2985–2992 [View Article][PubMed]
     [Google Scholar]
  11. Patel J. B. 2001; 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory. Mol Diagn 6:313–321[PubMed] [CrossRef]
     [Google Scholar]
  12. Reuben A. G., Musher D. M., Hamill R. J., Broucke I. 1989; Polymicrobial bacteremia: clinical and microbiologic patterns. Rev Infect Dis 11:161–183 [View Article][PubMed]
     [Google Scholar]
  13. Ronaghi M. 2001; Pyrosequencing sheds light on DNA sequencing. Genome Res 11:3–11 [View Article][PubMed]
     [Google Scholar]
  14. Ronaghi M., Elahi E. 2002; Pyrosequencing for microbial typing. J Chromatogr B Analyt Technol Biomed Life Sci 782:67–72 [View Article][PubMed]
     [Google Scholar]
  15. Ronaghi M., Karamohamed S., Pettersson B., Uhlén M., Nyrén P. 1996; Real-time DNA sequencing using detection of pyrophosphate release. Anal Biochem 242:84–89 [View Article][PubMed]
     [Google Scholar]
  16. Stager C. E., Davis J. R. 1992; Automated systems for identification of microorganisms. Clin Microbiol Rev 5:302–327[PubMed]
     [Google Scholar]
  17. Sundquist A., Bigdeli S., Jalili R., Druzin M. L., Waller S., Pullen K. M., El-Sayed Y. Y., Taslimi M. M., Batzoglou S., Ronaghi M. 2007; Bacterial flora-typing with targeted, chip-based pyrosequencing. BMC Microbiol 7:108 [View Article][PubMed]
     [Google Scholar]
  18. Tang Y. W., Ellis N. M., Hopkins M. K., Smith D. H., Dodge D. E., Persing D. H. 1998; Comparison of phenotypic and genotypic techniques for identification of unusual aerobic pathogenic Gram-negative bacilli. J Clin Microbiol 36:3674–3679[PubMed]
     [Google Scholar]
  19. Wang Q., Garrity G. M., Tiedje J. M., Cole J. R. 2007; Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267 [View Article][PubMed]
     [Google Scholar]
  20. Wimmer J. L., Long S. W., Cernoch P., Land G. A., Davis J. R., Musser J. M., Olsen R. J. 2012; Strategy for rapid identification and antibiotic susceptibility testing of Gram-negative bacteria directly recovered from positive blood cultures using the Bruker MALDI Biotyper and the BD Phoenix system. J Clin Microbiol 50:2452–2454 [View Article][PubMed]
     [Google Scholar]
  21. Woese C. R. 1987; Bacterial evolution. Microbiol Rev 51:221–271[PubMed]
     [Google Scholar]
  22. Wüppenhorst N., Consoir C., Lörch D., Schneider C. 2012; Direct identification of bacteria from charcoal-containing blood culture bottles using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Eur J Clin Microbiol Infect Dis (in press) [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.049163-0
Loading
Identification of bacteria directly from positive blood culture samples by DNA pyrosequencing of the 16S rRNA gene
J. Med. Microbiol. 61, 1556 (2012); https://doi.org/10.1099/jmm.0.049163-0
/content/journal/jmm/10.1099/jmm.0.049163-0
/content/journal/jmm/10.1099/jmm.0.049163-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

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