Rapid detection of CTX-M-producing Enterobacteriaceae in urine samples

Objectives: CTX-M extended-spectrum beta-lactamases (ESBLs) are emerging worldwide. Fast and reliable detection techniques may become mandatory for implementing proper treatment and infection control measures. Here, a bla(CTX-M)-Specific LightCycler real-time PCR (LC-PCR) assay based on hybridization probes was developed. Methods: Urine samples positive for Gram-negative bacilli as revealed by Gram staining were collected over a 3 month period at Bicetre hospital, France. Aliquots of these urine samples were frozen for subsequent molecular analysis, and the bacteria were cultured and identified by standard bacteriological techniques (biochemical tests, disc diffusion antibiogram and synergy testing). LC-PCR and standard PCR followed by sequencing was performed on all ESBL-positive and on 70 randomly chosen ESBL-negative urine samples. Results: Over the study period, 810 urine samples were collected from 655 patients. Thirty-six ESBL-producing Enterobacteriaceae, mostly Escherichia coli (77%), were identified from 29 patients, of which half were outpatients. Twenty-five urine samples (19 patients) were found to be positive for bla(CTX-M) genes using the LC-PCR assay. The bla(CTX-M) genes belonged to the bla(CTX-M-1), bla(CTX-M-9) and bla(CTX-M-2) groups (68%, 24% and 8%, respectively). Standard PCR and sequencing of the entire bla(CTX-M) genes confirmed the LC-PCR results; 17 CTX-M-15, 6 CTX-M-9 and 2 CTX-M-2. Among the remaining ESBLs, eight were of the TEM type and three of the SHV type. Conclusions: The LC-PCR assay represents a powerful tool for rapid identification of CTX-M producers in urine samples.