Open AccessDetection of gyrA Mutations in Quinolone-Resistant Salmonella enterica by Denaturing High-Performance Liquid Chromatography
Author(s) -
Deborah J. Eaves,
Ernesto Liébana,
Martin J. Woodward,
Laura J. V. Piddock
Publication year - 2002
Publication title -
journal of clinical microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.349
H-Index - 255
eISSN - 1070-633X
pISSN - 0095-1137
DOI - 10.1128/jcm.40.11.4121-4125.2002
Subject(s) - denaturing high performance liquid chromatography , biology , single strand conformation polymorphism , salmonella enterica , quinolone , salmonella , microbiology and biotechnology , genetics , dna gyrase , mutation , gene , escherichia coli , bacteria , antibiotics
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 ofgyrA fromSalmonella serovars. A total of 203 isolates ofSalmonella 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.