Phenotypic detection of extended-spectrum and AmpC β-lactamases by a new spot-inoculation method and modified three-dimensional extract test: Comparison with the conventional three-dimensional extract test

Abstract

Objectives: To develop an easy, rapid and reproducible spot-inoculation method for phenotypic detection of extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases and to make the existing three-dimensional extract test more convenient for use in routine diagnostic laboratories. Methods: ESBL and AmpC producing and non-producing isolates of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, as identified by the conventional three-dimensional extract test, were used to evaluate the modified procedures. Whole bacterial cells and freeze-thaw preparations, as β-lactamase sources, were strategically applied to culture plates near ceftazidime and cefoxitin discs on a lawn inoculum of E. coli ATCC 25922. Technical variations of the test included placing the β-lactamase-containing inoculum into slits, wells and trenches, or onto the surface as spots at varying distances from the discs, and adding clavulanate or cloxacillin to the three-dimensional inoculum to confirm the presence of ESBLs and AmpC β-lactamases, respectively. Results: All the methods adopted for ESBL and AmpC detection by using the whole bacterial cells gave positive results. However, the best results were given by the spot-inoculation method. In modifications using the enzymic extracts, the enhanced growth of surface organisms was better appreciated in the designed modifications compared with the conventional methods. Conclusions: The method described here is simple and cost-effective. Furthermore, up to 16 isolates may be tested on a single culture plate, thus it is a less labour-intensive and more economic technique than other reported phenotypic methods. © The British Society for Antimicrobial Chemotherapy 2004; all rights reserved.