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J Antimicrob Chemother. 2004 Sep;54(3):684-7. Epub 2004 Aug 4.

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

Author information

1
Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh-202002, Uttar Pradesh, India. shahidsahar@yahoo.co.in

Abstract

OBJECTIVES:

To develop an easy, rapid and reproducible spot-inoculation method for phenotypic detection of extended-spectrum beta-lactamases (ESBLs) and AmpC beta-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 beta-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 beta-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 beta-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.

PMID:
15294886
DOI:
10.1093/jac/dkh389
[Indexed for MEDLINE]

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