| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the Department of Pathology,
*
University of Pittsburgh, Pittsburgh; and Magee Women’s Research Institute,
Pittsburgh, Pennsylvania
Infants admitted to neonatal intensive care units for suspicion of bacterial sepsis receive at least two broad-spectrum antibiotics for a minimum of 48 to 72 hours to cover both gram-positive and gram-negative organisms while awaiting blood culture results. On average, bacterial growth becomes detectable within 12 to 24 hours, with an additional 24 to 48 hours required for identification. We have previously described using a 16S rRNA PCR assay for screening neonatal blood for bacterial DNA. Combining PCR with DNA sequencing could prove a faster means of detecting bacteria than culture-based identification. If successful, antibiotic therapy could be appropriately tailored sooner, thus sparing infants the administration of unnecessary antibiotics. Our goal was to assess the potential of pyrosequencing to differentiate between bacteria commonly associated with neonatal sepsis. To begin, full-length sequencing of the 380-bp 16S rRNA amplicons from representative bacteria was conducted (ABI 3100) and several databases queried. These included Staphylococcus sp., Streptococcus sp., Listeria sp., and numerous gram-negative rods. The sequences from clinical isolates were identical to those present in the published databases for the same bacteria. As a result, an informative 15 bases within the 380-bp amplicon was targeted for pyrosequencing following enrichment culture and PCR amplification. A total of 643 bacterial isolates commonly associated with neonatal sepsis, and 15 PCR-positive, culture-positive neonatal whole blood samples were analyzed by pyrosequencing. Results of DNA sequencing and culture identification were compared. In summary, we were successful at using PCR and pyrosequencing together to accurately differentiate between highly diverse bacterial groups.
This article has been cited by other articles:
 |
|
 |
|
  A. Huyghe, P. Francois, Y. Charbonnier, M. Tangomo-Bento, E.-J. Bonetti, B. J. Paster, I. Bolivar, D. Baratti-Mayer, D. Pittet, J. Schrenzel, et al. Novel Microarray Design Strategy To Study Complex Bacterial Communities Appl. Envir. Microbiol., March 15, 2008; 74(6): 1876 - 1885. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Luna, L. R. Fasciano, S. C. Jones, B. L. Boyanton Jr., T. T. Ton, and J. Versalovic DNA Pyrosequencing-Based Bacterial Pathogen Identification in a Pediatric Hospital Setting J. Clin. Microbiol., September 1, 2007; 45(9): 2985 - 2992. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Dejeux, V. Audard, C. Cavard, I. G. Gut, B. Terris, and J. Tost Rapid Identification of Promoter Hypermethylation in Hepatocellular Carcinoma by Pyrosequencing of Etiologically Homogeneous Sample Pools J. Mol. Diagn., September 1, 2007; 9(4): 510 - 520. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Jordan, M. B. Durso, A. R. Butchko, J. G. Jones, and B. S. Brozanski Evaluating the Near-Term Infant for Early Onset Sepsis: Progress and Challenges to Consider with 16S rDNA Polymerase Chain Reaction Testing J. Mol. Diagn., July 1, 2006; 8(3): 357 - 363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Jordan and M. B. Durso Real-Time Polymerase Chain Reaction for Detecting Bacterial DNA Directly from Blood of Neonates Being Evaluated for Sepsis J. Mol. Diagn., November 1, 2005; 7(5): 575 - 581. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ogino, T. Kawasaki, M. Brahmandam, L. Yan, M. Cantor, C. Namgyal, M. Mino-Kenudson, G. Y. Lauwers, M. Loda, and C. S. Fuchs Sensitive Sequencing Method for KRAS Mutation Detection by Pyrosequencing J. Mol. Diagn., August 1, 2005; 7(3): 413 - 421. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
