Using newly developed multiplex polymerase chain reaction and melting curve analysis for detection and discrimination of β-lactamases in Escherichia coli isolates from intensive care patients

A total of 78 bacterial strains with known beta-lactamases were used to optimize a rapid detection system consisting of multiplex polymerase chain reaction and melting curve analysis to amplify and identify blaTEM, blaSHV, and blaCTX-M genes in a single reaction. Additionally, to evaluate the applicability of this method, 32 clinical isolates of Escherichia coli displaying an extended-spectrum beta-lactamase phenotype from patients hospitalized at intensive care units were tested. Results were analyzed by the Rotor-Gene operating software and Rotor-Gene ScreenClust HRM Software. The individual melting curves differed by a temperature shift or curve shape, according to the presence of beta-lactamase genes. With the use of this method and direct sequencing, blaCTX-M-15-like was identified as the most prevalent beta-lactamase gene. In conclusion, this novel detection system seems to be a suitable tool for rapid detection of present beta-lactarnase genes and their characterization. (C) 2011 Elsevier Inc. All rights reserved.