JAC Advance Access originally published online on August 4, 2004
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Journal of Antimicrobial Chemotherapy 2004 54(3):684-687; doi:10.1093/jac/dkh389
JAC vol.54 no.3 © The British Society for Antimicrobial Chemotherapy 2004; all rights reserved.
Phenotypic detection of extended-spectrum and AmpC ß-lactamases by a new spot-inoculation method and modified three-dimensional extract test: comparison with the conventional three-dimensional extract test
Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh—202002, Uttar Pradesh, India
* Corresponding author. Tel: +91-571-2720382; Fax: +91-571-2721776; Email: shahidsahar{at}yahoo.co.in
Objectives: To develop an easy, rapid and reproducible spot-inoculation method for phenotypic detection of extended-spectrum ß-lactamases (ESBLs) and AmpC ß-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 ß-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 ß-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 ß-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.
Keywords: direct-spot test , well-extract test , modified enzymic extraction , modifications , user-friendly method
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