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JAC Advance Access originally published online on January 20, 2009
Journal of Antimicrobial Chemotherapy 2009 63(3):497-501; doi:10.1093/jac/dkn535
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© The Author 2009. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

A simple disc diffusion method for detecting AmpC and extended-spectrum β-lactamases in clinical isolates of Enterobacteriaceae

Helen Derbyshire1, Gemma Kay2, Katie Evans3, Carmel Vaughan3, Umadevi Kavuri4 and Trevor Winstanley1,*

1 Department of Microbiology, Royal Hallamshire Hospital, Glossop Road, Sheffield S10 2JF, UK 2 Biomedical Research Centre, Faculty of Health and Wellbeing, Sheffield Hallam University, Sheffield S1 1WB, UK 3 Mast Diagnostics, Mast House, Derby Road, Bootle L20 1EA, UK 4 School of Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK

Received 27 October 2008; returned 11 December 2008; revised 15 December 2008; accepted 15 December 2008

* Corresponding author. Tel: +44-114-271-3126; Fax: +44-114-278-9376; E-mail: trevor.winstanley{at}sth.nhs.uk

Background: We sought to determine whether extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases (derepressed and inducible), alone and in combination, could be detected in unidentified members of the Enterobacteriaceae using a simple, overnight disc diffusion test.

Methods: The genetic basis of antibiotic resistance in cephalosporin-resistant wild-type (n = 140) and culture collection (n = 140) isolates of Enterobacteriaceae was determined using PCR. A scheme for detecting these resistance mechanisms phenotypically was devised using five antibiotic discs: cefpodoxime ± clavulanate; cefepime ± clavulanate and cefoxitin.

Results and conclusions: AmpC β-lactamases (derepressed and inducible) and ESBLs, alone and in combination, could reliably be detected using a disc diffusion method. ESBLs alone could be detected on the basis of a difference of >5 mm between cefpodoxime/clavulanate and cefpodoxime (10 µg) discs. ESBLs, in the presence of AmpC β-lactamases, could be detected using a difference of >5 mm between cefepime/clavulanate and cefepime (30 µg) discs. AmpC β-lactamases could be detected using a difference of >14 mm between cefepime/clavulanate and cefpodoxime/clavulanate discs. Inducible AmpC β-lactamases could be discerned by observing the blunting of the cefpodoxime or cefpodoxime/clavulanate zones in proximity to cefoxitin (30 µg) discs.

Keywords: ESBLs , resistance phenotypes , mechanisms of resistance


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