Identification of CTX-M-Type Extended-Spectrum-β-Lactamase Genes Using Real-Time PCR and Pyrosequencing

Abstract

CTX-M extended-spectrum ␤-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 ESBLproducing 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 ␤-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.

Variation-Tolerant Capture and Multiplex Detection of Nucleic Acids: Application to Detection of Microbes

“…2). Further, different Escherichia coli and Klebsiella strains harboring antibiotic resistance genes belonging to CTX-M groups I or IV (14) were accurately detected. Conclusively, the 22-plex VOCMA test showed high specificity (Fig.…”

Class 1 and 2 integrons, sul resistance genes and antibiotic resistance in Escherichia coli isolated from Dongjiang River, South China

“…A few colonies of each strain were suspended in 100 ml distilled water and heated at 100 C for 20 min, followed by immediately cooling on ice for 10 min. The suspension was centrifuged at 16,000 g for 10 min and the supernatant was stored at À20 C for PCR assays (Cattoir et al, 2007;Gueimonde et al, 2004;Naas et al, 2007).…”

Evaluation of a DNA Microarray (Check-MDR CT102) for Rapid Detection of TEM, SHV, and CTX-M Extended-Spectrum β-Lactamases and of KPC, OXA-48, VIM, IMP, and NDM-1 Carbapenemases

“…The vast majority of ESBLs belong to the TEM-, SHV-and CTX-M-type enzymes (15,20,24). While TEM-and SHV-type ESBLs arise via substitutions in strategically positioned amino acids (e.g., Gly238 and Arg164) from the natural narrow-spectrum TEM-1, TEM-2, or SHV-1 ␤-lactamase genes, all known CTX-M enzymes demonstrate expanded-spectrum activity (19,24). Since ESBL-producing bacteria are often multidrug resistant (MDR), carbapenems represent one of the therapeutic options of last resort for life-threatening infections due to these organisms.…”

“…However, routine phenotypic testing may fail to identify ESBL producers, and it also usually delays the final report by an additional 24 h (1). Several molecular assays such as real-time PCR targeting ESBL genes have been proposed for the detection of CTX-M-type ESBLs (2,19,23,25,34), but these techniques are not suited for the rapid detection of TEM and SHV ESBLs in (daily) routine use, since sequencing is required to distinguishing ESBLs from the non-ESBL variants (18,27).…”