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JAC Advance Access originally published online on January 25, 2007
Journal of Antimicrobial Chemotherapy 2007 59(3):387-395; doi:10.1093/jac/dkl505
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© The Author 2007. 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

A novel reverse-line hybridization assay for identifying genotypes of CTX-M-type extended-spectrum ß-lactamases

V. M. Ensor1,2,*, D. M. Livermore3 and P. M. Hawkey1,2

1 West Midlands Health Protection Agency, Heart of England NHS Foundation Trust, Bordesley Green, Birmingham B9 5SS, UK 2 Antimicrobial Agents Research Group, Division of Immunity and Infection, Institute of Biomedical Research, University of Birmingham, Birmingham B15 2TT, UK 3 Antibiotic Resistance Monitoring and Reference Laboratory, Centre for Infections, Health Protection Agency, Colindale, London NW9 5EQ, UK

Received 26 October 2006; returned 13 November 2006; revised 16 November 2006; accepted 17 November 2006

* Corresponding author. Tel: +44-121-424-1240; Fax: +44-121-772-6229; E-mail: vicki.ensor{at}heartofengland.nhs.uk

Objectives: To develop a reverse-line hybridization assay to identify CTX-M genotypes, potentially useful for large-scale investigation of surveillance collections.

Methods: Isolates carrying previously characterized blaCTX-M genes were used to develop the method. In addition, 334 isolates from five separate surveys were used to validate the method. CTX-M group was known from an independent multiplex PCR for 122 isolates and genotype was confirmed for 80 isolates by DNA sequencing. A multiplex PCR was designed to amplify a genotype-specific region within the blaCTX-M open-reading frame. Oligonucleotides were designed to hybridize to regions within each amplicon, covering mutations that distinguish among blaCTX-M genotypes.

Results: CTX-M phylogenetic groups were identified by the multiplex PCR with 100% concordance. The reverse-line hybridization assay specifically identified commonly-reported variants within these groups (98.7% concordance).

Conclusions: The hybridization method enabled precise identification of CTX-M genes, rather than just to group level, without the need for DNA sequencing. In its present format, the method enables 43 isolates to be processed per membrane, giving results within one working day. It is a useful tool for the epidemiological investigation of blaCTX-M genes among survey collections of Enterobacteriaceae.

Keywords: ESBLs , genotyping , multiplex PCR , molecular epidemiology


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