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Antimicrob. Agents Chemother. doi:10.1128/AAC.00611-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Identification of CTX-M-Type Extended-Spectrum ß-Lactamases genes based on Real Time PCR and Pyrosequencing

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

^* To whom correspondence should be addressed. Email: thierry.naas{at}bct.ap-hop-paris.fr,

	Abstract

CTX-M extended-spectrum ß-lactamases (ESBLs) are increasingly prevalent worldwide among Escherichia coli mostly in community-acquired urinary tract infections. Fast and reliable technique for identification of CTX-M-enzymes is becoming a challenge for the microbiology laboratory. A fast real-time PCR amplification, using degenerated primers specific for all the bla_CTX-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 sub-groups. Phylogenetic trees constructed with the entire bla_CTX-M genes and with both pyrosequenced regions (29 bp) gave similar results, suggesting a powerful discriminatory ability of this technique termed real-time detection and sequencing method (RTDSM). 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, 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%) 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.

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