title: Identification of CTX-M-type extended-spectrum-beta-lactamase genes using real-time PCR and pyrosequencing.
abstract: CTX-M extended-spectrum beta-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 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 subgroups. Phylogenetic trees constructed with the entire bla(CTX-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 beta-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.
authors.affiliation: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre cedex, France. thierry.naas@bct.ap-hop-paris.fr
authors.forename: Thierry
authors.initials: T
authors.lastname: Naas
authors.forename: Cynthia
authors.initials: C
authors.lastname: Oxacelay
authors.forename: Patrice
authors.initials: P
authors.lastname: Nordmann
mesh_terms: D001483:Base Sequence; D016000:Cluster Analysis; D017714:Community-Acquired Infections / Q000382:microbiology; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D004926:Escherichia coli / Q000235:genetics; D004927:Escherichia coli Infections / Q000382:microbiology; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D018440:beta-Lactam Resistance / Q000235:genetics; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D001618:beta-Lactamases