Identification of CTX-M-type extended-spectrum-β-lactamase genes using real-time PCR and pyrosequencing

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 Bicetre 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.