This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Naas, T.
Right arrow Articles by Nordmann, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Naas, T.
Right arrow Articles by Nordmann, P.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, January 2007, p. 223-230, Vol. 51, No. 1
0066-4804/07/$08.00+0     doi:10.1128/AAC.00611-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of CTX-M-Type Extended-Spectrum-ß-Lactamase Genes Using Real-Time PCR and Pyrosequencing{triangledown}

Thierry Naas,* Cynthia Oxacelay, and Patrice Nordmann

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI, 94275 Le Kremlin-Bicêtre, France

Received 18 May 2006/ Returned for modification 21 July 2006/ Accepted 23 October 2006

CTX-M extended-spectrum ß-lactamases (ESBLs) are increasingly prevalent worldwide among Escherichia coli bacteria, mostly in community-acquired urinary tract infections. Finding a fast and reliable technique for identification of CTX-M enzymes is becoming a challenge for the microbiology laboratory. A fast real-time PCR amplification technique, using degenerated primers specific for all the blaCTX-M alleles, coupled to real-time pyrosequencing was developed. The five CTX-M groups were unambiguously identified by pyrosequencing a 13-bp DNA region. Further sequencing of an additional 16-bp region allowed further division into subgroups. Phylogenetic trees constructed with the entire blaCTX-M genes and with both pyrosequenced regions (29 bp) gave similar results, suggesting that this technique, termed the real-time detection and sequencing method, has a powerful discriminatory ability. This high-throughput technique has been evaluated by screening 48 ESBL-producing E. coli isolates recovered from the Bicêtre hospital (France) in 2004. Forty-four of these strains were CTX-M positive by real-time PCR detection and direct pyrosequencing of the PCR products, which identified CTX-M-15 as the main CTX-M-type ß-lactamase. Pulsed-field gel electrophoresis analysis of these strains revealed that several clones, of which one CTX-M-15-positive clone was predominant (60%), were identified both in nosocomial and in community-acquired isolates. The combination of real-time PCR with pyrosequencing represents a powerful tool for epidemiological studies of CTX-M producers. This assay has the potential to be used in a diagnostic laboratory since up to 96 bacterial isolates may be screened in less than 3 h.

* Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre cedex, France. Phone: 33-1-45-21-29-86. Fax: 33-1-45-21-63-40. E-mail: thierry.naas{at}bct.ap-hop-paris.fr.

{triangledown} Published ahead of print on 6 November 2006.

Antimicrobial Agents and Chemotherapy, January 2007, p. 223-230, Vol. 51, No. 1
0066-4804/07/$08.00+0     doi:10.1128/AAC.00611-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Jones, C. H., Ruzin, A., Tuckman, M., Visalli, M. A., Petersen, P. J., Bradford, P. A. (2009). Pyrosequencing Using the Single-Nucleotide Polymorphism Protocol for Rapid Determination of TEM- and SHV-Type Extended-Spectrum {beta}-Lactamases in Clinical Isolates and Identification of the Novel {beta}-Lactamase Genes blaSHV-48, blaSHV-105, and blaTEM-155. Antimicrob. Agents Chemother. 53: 977-986 [Abstract] [Full Text]  
  • Haanpera, M., Forssten, S. D., Huovinen, P., Jalava, J. (2008). Typing of SHV Extended-Spectrum {beta}-Lactamases by Pyrosequencing in Klebsiella pneumoniae Strains with Chromosomal SHV {beta}-Lactamase. Antimicrob. Agents Chemother. 52: 2632-2635 [Abstract] [Full Text]  
  • Reglier-Poupet, H., Naas, T., Carrer, A., Cady, A., Adam, J.-M., Fortineau, N., Poyart, C., Nordmann, P. (2008). Performance of chromID ESBL, a chromogenic medium for detection of Enterobacteriaceae producing extended-spectrum {beta}-lactamases. J Med Microbiol 57: 310-315 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»