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Journal of Clinical Microbiology, November 2002, p. 4121-4125, Vol. 40, No. 11
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.11.4121-4125.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Detection of gyrA Mutations in Quinolone-Resistant Salmonella enterica by Denaturing High-Performance Liquid Chromatography

Deborah J. Eaves,1 Ernesto Liebana,2 Martin J. Woodward,2 and Laura J. V. Piddock1*

Antimicrobial Agents Research Group, Division of Infection and Immunity, University of Birmingham, Birmingham B15 2TT,1 Department of Bacterial Diseases, Veterinary Laboratories Agency, New Haw, Surrey KT15 3NB, United Kingdom2

Received 24 April 2002/ Returned for modification 13 July 2002/ Accepted 20 August 2002

Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a rapid screening and identification method for DNA sequence variation detection in the quinolone resistance-determining region of gyrA from Salmonella serovars. A total of 203 isolates of Salmonella were screened using this method. DHPLC analysis of 14 isolates representing each type of novel or multiple mutations and the wild type were compared with LightCycler-based PCR-gyrA hybridization mutation assay (GAMA) and single-strand conformational polymorphism (SSCP) analyses. The 14 isolates gave seven different SSCP patterns, and LightCycler detected four different mutations. DHPLC detected 11 DNA sequence variants at eight different codons, including those detected by LightCycler or SSCP. One of these mutations was silent. Five isolates contained multiple mutations, and four of these could be distinguished from the composite sequence variants by their DHPLC profile. Seven novel mutations were identified at five different loci not previously described in quinolone-resistant salmonella. DHPLC analysis proved advantageous for the detection of novel and multiple mutations. DHPLC also provides a rapid, high-throughput alternative to LightCycler and SSCP for screening frequently occurring mutations.

* Corresponding author. Mailing address: Antimicrobial Agents Research Group, Division of Infection and Immunity, University of Birmingham, Birmingham B15 2TT, United Kingdom. Phone: (44) 121-414-6966. Fax: (44) 121-414-3454. E-mail: l.j.v.piddock{at}bham.ac.uk.

Journal of Clinical Microbiology, November 2002, p. 4121-4125, Vol. 40, No. 11
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.11.4121-4125.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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